Jane E.

Sykes, BVSc, PhD, DACVIM

Professor
Department of Medicine and Epidemiology
University of California
Davis, California
3251 Riverport Lane
St. Louis, Missouri 63043

CANINE AND FELINE INFECTIOUS DISEASES ISBN: 978-1-4377-0795-3

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Library of Congress Cataloging-in-Publication Data

Sykes, Jane E.
Canine and feline infectious diseases / Jane E. Sykes.
p. ; cm.
Includes bibliographical references and index.
ISBN 978-1-4377-0795-3 (hard back)
I. Title.
[DNLM: 1. Communicable Diseases--veterinary. 2. Cat Diseases. 3. Dog Diseases. SF 781]
SF781
636.089’69--dc23 2013010391

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 To Terry Nagle, who did countless loads of laundry and cooked
children’s meals while I worked on this book. And to my children,
Henry, Bridget, and Gillian, who through looking over my shoulder
have learned a little about infectious diseases.

Perhaps readers might enjoy seeing some

of what they have learned:

Cryptococcus Antibodies, Phagocytes and Microbes

Dermatophytes Flagellae Virus

Virus Poxvirus Amoeba

Preface
The goal of this first edition of Canine and Feline Infectious
Diseases was to provide a text for veterinary students and cli-
nicians with a strong clinical emphasis. Authorship has been
limited to veterinarians who have had direct and considerable
clinical experience with diagnosis and treatment of the diseases
described. The chapters have been presented in a consistent and
logical format with extensive use of tables, photographs, and
line drawings, so that veterinarians and students can quickly
find answers to questions they have that relate to infectious dis-
eases in dogs and cats.
Organization
This book contains two parts and eight sections. The first
part, Basic Principles in the Diagnosis and Management of
Small Animal Infection, contains three sections: Laboratory
Diagnosis of Infectious Diseases, Antiinfective Therapy, and
Basic Principles of Infection Control. The second part, Major
Infectious Diseases and their Etiologic Agents, systematically
discusses specific infectious diseases of dogs and cats in a way
that is practical and relevant to the veterinary clinician.
The first section, Laboratory Diagnosis of Canine and
Feline Infectious Diseases, is co-authored by a small animal
clinician and a clinical microbiologist. It aims to help practic-
ing veterinarians select the best type of test for detection of
an infectious disease, collect the most appropriate specimens
for testing, optimally transport specimens to the laboratory,
and most importantly, to interpret test results properly. The
second section, Antiinfective Therapy, is co-authored by a
clinician and a veterinary pharmacologist. The goal of this
section is to help veterinarians use first principles to select
the most appropriate drug for treatment of a suspected or
confirmed infectious disease, understand the mechanisms
of action and adverse effects of various antimicrobial drugs
used in practice, and utilize treatment regimes that minimize
development of antimicrobial resistance. The third section,
Basic Principles for Infection Control, is co-authored by
veterinary clinicians who have extensive practical experi-
ence with infectious disease control in veterinary hospitals.
Practical protocols for the management of canine and feline
patients suspected to have infectious diseases are provided,
including handling, disinfection, isolation, and vaccination
protocols.
The second half of this book addresses specific infectious
diseases of dogs and cats, which are grouped into viral, bacte-
rial, fungal, and protozoal diseases (Sections 1 through 4) as
well as clinical syndromes (Section 5). A summary box at the
beginning of the chapter provides an overview of the disease or
diseases covered. Each chapter contains information on etiology
and epidemiology, clinical signs and their pathogenesis; physical
examination abnormalities, diagnosis, treatment and prognosis,
immunity and vaccination, prevention, and public health impli-
cations. Some information on the equivalent disease in humans
is provided whenever pertinent. Where relevant, maps and life
cycle drawings are included, and chapters are illustrated with
photographs of case material. All chapters contain a table that
summarizes the performance, advantages and disadvantages of
the major diagnostic assays available for each disease, and many
chapters also contain a table that provides antimicrobial drug
treatment recommendations and dosing. Key statements in the
text are referenced in case the reader desires additional back-
ground information, although provision of an exhaustive list
of references is not in the scope of this book. A few suggested
readings are provided.
One (and occasionally two) case example(s) can be found in
a standard format at the end of almost all of the chapters in the
second half of the book. These cases are designed to empha-
size key epidemiological and clinical aspects of the disease, the
application of diagnostic assays, treatment, and prognosis. The
cases were seen at the authors’ institutions, and many include
the results of full laboratory assessments, radiology, cytol-
ogy, and histopathology reports. Unless otherwise specified,
all urinalyses and urine cultures were performed on specimens
collected by cystocentesis, and all thoracic radiographic stud-
ies consisted of left and right lateral and ventrodorsal views.
Abdominal ultrasound examinations consisted of complete
studies of intraabdominal structures. Because these are “real-
world” examples, diagnosis, management, and outcomes are
not always perfect or ideal. Nevertheless, this can provide
insight into real-life problems that may confound accurate
diagnosis and management of the disease, such as misdiag-
nosis when an infectious disease resembles other infectious or
non-infectious diseases on the differential diagnosis list. Other
problems illustrated include limitations of the sensitivity or
specificity of diagnostic tests used, or hurdles imposed by the
availability of client financial resources. A comment section at
the end of the case provides the author(s’) perspective on the
case. It should be recognized that infectious diseases can vary
widely in their clinical presentation and response to treatment,
so each case just provides one “view” of the infectious disease
described in the chapter.
Jane E. Sykes
vii
Acknowledgments
I am incredibly grateful to the huge number of veterinarians,
residents, and veterinary students worldwide who have encour-
aged me to write and edit this book. I would particularly like to
acknowledge all of my colleagues at the University of ­California,
Davis, who over the years provided a large number of the care-
fully collected and valuable images in this book. Special men-
tions go to Barbara Byrne, Patricia Pesavento, and Eileen Samitz
who spent much additional time to prepare or provide material
for photography for this book.
I would like to acknowledge the illustrator for the book, Ted
Huff, who has performed the amazing task of converting a large
viii
number of complicated maps and life cycle drawings into crisp,
clean diagrams that are easy to understand and remember. I
have also been fortunate to work with a phenomenal team at
Elsevier who were consistently encouraging, efficient, and made
every effort to be accommodating. I would like to thank my
internal medicine and infectious disease colleagues worldwide,
many of whom contributed to chapters for this book, for inspir-
ing me to have a passion for the study of small animal internal
medicine and infectious diseases. Last but not least I would like
to acknowledge my family, who cheered me on the whole way.
Jane E. Sykes
Contributors

Gad Baneth, DVM, PhD, DECVCP Leah A. Cohn, DVM, PhD, DACVIM
Professor of Veterinary Medicine Professor, Small Animal Internal Medicine
The Robert H. Smith Faculty of Agriculture, Food and Department of Veterinary Medicine and Surgery
Environment College of Veterinary Medicine
Koret School of Veterinary Medicine University of Missouri
The Hebrew University of Jerusalem Columbia, Missouri
Rehovot, Israel Cytauxzoonosis
Leishmaniosis
Sarah D. Cramer, DVM, DACVP
Stephen C. Barr, BVSc, MVS, PhD, DACVIM Cancer Research Training Fellow
Professor of Medicine Comparative Biomedical Scientist Training Program
Chief, Section of Small Animal Medicine National Cancer Institute
College of Veterinary Medicine National Institutes of Health
Cornell University Bethesda, Maryland
Ithaca, New York Pseudorabies
Trypanosomiasis
Autumn P. Davidson, DVM, MS, DACVIM
Malcolm Bennett, BVSc, PhD, MRCVS, FRCPath Clinical Professor
Professor of Veterinary Pathology, Infection, and Global Department of Medicine and Epidemiology
Health School of Veterinary Medicine
Department of Veterinary Pathology University of California
University of Liverpool Davis, California;
Liverpool, United Kingdom Staff Internist
Feline Poxvirus Infections Internal Medicine/Reproduction
PetCare Veterinary Hospital
Adam J. Birkenheuer, DVM, DACVIM, PhD Santa Rosa, California
Associate Professor Canine Herpesvirus Infection
Department of Clinical Sciences Canine Brucellosis
College of Veterinary Medicine
North Carolina University Steven Epstein, DVM, DACVECC
Raleigh, North Carolina Assistant Professor of Clinical Small Animal Emergency and
Babesiosis Critical Care
Department of Veterinary Surgical and Radiological Sciences
Edward B. Breitschwerdt, DVM, DACVIM School of Veterinary Medicine
Professor of Medicine and Infectious Diseases University of California
Department of Clinical Sciences Davis, California
College of Veterinary Medicine Infections of the Cardiovascular System
North Carolina State University
Raleigh, North Carolina Janet E. Foley, DVM, PhD
Rocky Mountain Spotted Fever Professor
Department of Veterinary Medicine and Epidemiology
Bruno B. Chomel, DVM, PhD School of Veterinary Medicine
Professor University of California
Population Health & Reproduction Davis, California
University of California Anaplasmosis
Davis, California
Rabies
Bartonellosis
Yersinia pestis (Plague) and Other Yersinioses
Tularemia

iii
iv CONTRIBUTORS

Craig E. Greene, DVM, MS, DACVIM Remo Lobetti, BVSc, MMedVet (Med), PhD, DECVIM
Professor Emeritus and Josiah Meigs Distinguished Teaching (Internal Medicine)
Professor Internist
Departments of Infectious Diseases and Small Animal Medicine Bryanston Veterinary Hospital
College of Veterinary Medicine Bryanston, South Africa
The University of Georgia Pneumocystosis
Athens, Georgia
Bacterial Meningitis Jennifer A. Luff, VMD, DACVP
Veterinary Medicine
Amy M. Grooters, DVM, DACVIM Department of Pathology, Microbiology, and Immunology
Professor, Companion Animal Medicine School of Veterinary Medicine
Department of Veterinary Clinical Sciences University of California
School of Veterinary Medicine Davis, California
Louisiana State University Viral Papillomatosis
Baton Rouge, Louisiana
Miscellaneous Fungal Diseases Richard Malik, DVSc, DipVetAn, MVetClinStud, PhD,
Pythiosis, Lagenidiosis, and Zygomycosis FACVSc, FASM
Valentine Charlton Veterinary Specialist
Danièlle A. Gunn-Moore, BSc, BVM&S, PhD, MACVSc, Centre for Veterinary Education
MRCVS, RCVS University of Sydney
Professor of Feline Medicine Sydney, Australia
Head of Companion Animal Sciences Cryptococcosis
Royal (Dick) School of Veterinary Studies
The University of Edinburgh Stanley L. Marks, BVSc, PhD, DACVIM (Internal Medicine,
Easter Bush, Midlothian, United Kingdom Oncology), DACVN
Mycobacterial Infections Professor
Department of Medicine and Epidemiology School of
Katrin Hartmann, Dr.med.vet, DMVH, DECVIM-CA Veterinary Medicine
Professor University of California
Center of Clinical Veterinary Sciences Davis, California
Clinic of Small Animal Medicine Salmonellosis
Ludwig-Maximilians-Universität, München Enteric Escherichia coli Infections
Munich, Germany Campylobacteriosis
Feline Leukemia Virus Infection Enteric Clostridial Infections
Gastric Helicobacter-like Infections
Amy S. Kapatkin, DVM, MS
Associate Professor of Orthopedic Surgery Lindsay K. Merkel, DVM, DACVIM
Department of Surgical and Radiological Sciences Internal Medicine Specialist
School of Veterinary Medicine Veterinary Medical Center
University of California College of Veterinary Medicine
Davis, California University of Minnesota
Osteomyelitis, Discospondylitis, and Infectious Arthritis St. Paul, Minnesota
Blastomycosis
Linda Kidd, DVM, PhD, DACVIM
Assistant Professor Terry M. Nagle, BVSc, MACVSc, DACVD
Department of Small Animal Internal Medicine Northern California Veterinary Specialist
College of Veterinary Medicine Emergency & Referral Center
Western University of Health Sciences Sacramento, California
Pomona, California Malassezia Infections
Rocky Mountain Spotted Fever Pyoderma, Otitis Externa, and Otitis Media

Michael R. Lappin, DVM, PhD, DACVIM Jacqueline M. Norris, BVSc, MVS, PhD
Professor of Infectious Disease Associate Professor of Veterinary Microbiology
Department of Clinical Sciences Faculty of Veterinary Science
College of Veterinary Medicine and Biomedical Sciences University of Sydney
Colorado State University Sydney, Australia
Fort Collins, Colorado Coxiellosis and Q Fever
Toxoplasmosis
Giardiasis
Trichomoniasis
Cryptosporidiosis
Isosporiasis
 CONTRIBUTORS v

Catherine A. Outerbridge, MVSc, DVM Séverine Tasker, BSc, BVSc, PhD, PGCert (HE), DSAM,
Associate Professor of Dermatology DECVIM-CA, MRCVS
Department of Medicine and Epidemiology Senior Lecturer in Small Animal Medicine
School of Veterinary Medicine School of Veterinary Sciences
University of California University of Bristol
Davis, California Bristol, Avon, United Kingdom
Dermatophytosis Hemoplasma Infections

Mark G. Papich, DVM, MS, DACVCP Nancy Vincent-Johnson, DVM, MS, DACVIM (SAIM),
Professor DACVPM
Department of Molecular Biomedical Sciences Veterinary Medical Officer
College of Veterinary Medicine Chief Clinical Services
North Carolina State University Fort Belvoir Veterinary Treatment Facility
Raleigh, North Carolina United States Army Veterinary Corps
Principles of Antiinfective Therapy Fort Belvoir, Virginia
Antiviral and Immunomodulatory Drugs Canine and Feline Hepatozoonosis
Antibacterial Drugs
Antifungal Drugs J. Scott Weese, DVM, DVSc, DACVIM
Antiprotozoal Drugs Professor
Department of Clinical Studies (Pathobiology)
Christine A. Petersen, DVM, PhD University of Guelph
Associate Professor in Veterinary Pathology Guelph, Ontario
Petersen and Jones Laboratory Infection Control Programs for Dogs and Cats
College of Veterinary Medicine
Iowa State University Jodi L. Westropp, DVM, PhD, DACVIM
Ames, Iowa Associate Professor
Leishmaniosis Department of Medicine and Epidemiology
School of Veterinary Medicine
Shelley C. Rankin, BSc (Hons), PhD University of California
Associate Professor (CE) of Microbiology Davis, California
New Bolton Center Bacterial Infections of the Genitourinary Tract
School of Veterinary Medicine
University of Pennsylvania Stephen D. White, DVM, DACVD
Philadelphia, Pennsylvania Professor
Isolation in Cell Culture Department of Medicine and Epidemiology
Immunoassays School of Veterinary Medicine
Isolation and Identification of Aerobic and Anaerobic Bacteria University of California
Isolation and Identification of Fungi Davis, California
Malassezia Infections
Ashley B. Saunders, DVM, DACVIM (Cardiology) Pyoderma, Otitis Externa, and Otitis Media
Assistant Professor
Department of Small Animal Clinical Sciences
College of Veterinary Medicine and Biomedical Sciences
Texas A&M University
College Station, Texas
Trypanosomiasis

Joseph Taboada, DVM, DACVIM (Internal Medicine)

Professor and Associate Dean
Department of Veterinary Clinical Sciences School of
Veterinary Medicine
Louisiana State University
Baton Rouge, Lousiana
Histoplasmosis
 SECTION 1
Laboratory Diagnosis of Canine
and Feline Infectious Diseases
Jane E. Sykes  and  Shelley C. Rankin
CHAPTER 1
Isolation in Cell Culture
Jane E. Sykes and Shelley C. Rankin
w KEY POINTS
• W  ith the increasing availability of nucleic acid–based testing,
cell culture is decreasingly used for diagnosis of infections
caused by obligate intracellular pathogens in dogs and cats.
• Cell culture remains an important technique for (a) confirma-
tion of a diagnosis when the results of molecular testing or
serology are unavailable or equivocal; (b) pathogen discovery;
and (c) vaccine manufacture. For some pathogens, cell cul-
ture is the most sensitive and specific method for organism culture contamination with bacteria or fungi.
detection.
• Before collection of specimens, veterinary clinicians should
communicate with the laboratory that is to perform the culture
to discuss the patient signalment, history, immune status, travel
history, nature of the suspected infection, and number of ani-
mals affected.
INTRODUCTION
Cell culture refers to the culture of nucleated (eukaryotic) cells
under controlled conditions within the laboratory. Infectious
agents that require living host cells for replication can only be
isolated in cell culture. With the advent of molecular diagnostic
assays based on nucleic acid detection, cell culture is being used
less often for routine clinical diagnostic purposes, because of the
long turnaround times (days to weeks), cost, and requirement for
significant technical expertise to perform cell culture and interpret
results (Table 1-1). Nevertheless, isolation of viral and intracellu-
lar bacterial and protozoal pathogens in cell culture remains an
important technique for the discovery of new pathogens, iden-
tification of organisms involved in disease when the results of
molecular testing or serology are unavailable or equivocal, the
propagation of isolates for research purposes, the generation of
organisms for vaccination purposes, and the establishment of
the efficacy of novel antimicrobial drugs. Vaccines for dogs and
cats that are propagated in cell culture include those for canine
2
• S pecimens are inoculated onto monolayers, and the infecting
organism is identified based on the presence of characteristic
cytopathic effect after a predictable incubation period, with or
without confirmatory antigen staining, electron microscopy, or
nucleic acid testing.
• False-negative results may occur as a result of inadequate spec-
imen collection, deterioration of organisms during transport, or
• Positive results do not imply that the organism detected is the
cause of an animal’s signs, because some organisms can be
present without causing disease. This is especially the case for
animals with respiratory or gastrointestinal disease.
distemper, canine adenovirus infections, parvovirus infections,
rabies, and feline viral and chlamydial respiratory tract disease.
Veterinary clinicians should remain aware of situations where cell
culture may be the best technique to identify the presence of an
infectious agent and the optimum methods for collection and
submission of specimens. Knowledge of cell culture methods can
help veterinary clinicians to submit the optimum specimens and
to understand laboratory turnaround times, potential complica-
tions, and how to interpret results.
Specimen Collection and Transport
Although cell culture can be used to propagate intracellular
bacteria and protozoa, it is most often used by clinicians for
the diagnosis of viral infections. Active communication between
the clinician and the laboratory that performs viral isolation is
recommended. Successful detection of viruses is highly depen-
dent on (a) collecting the appropriate specimens, (b) the tim-
ing of specimen collection, and (c) rapid and proper specimen
 CHAPTER 1  Isolation in Cell Culture 3

TABLE 1-1 BOX 1-1

Alternatives to Cell Culture for Diagnosis of Obligate Factors That Should Be Discussed with the Laboratory
Intracellular Pathogens That Infect Dogs and Cats before Collection of Specimens for Pathogen Isolation in
Cell Culture
System Affected and Most Other Diagnostic Tests
Common Agents Available
Patient species, breed, age, and environment
Respiratory Tract Number of animals affected
Canine respiratory coronavirus RT-PCR History and clinical signs
Canine adenovirus-2 PCR Immune status of the patient
Influenza viruses RT-PCR, antibody Geographic location and travel history
Canine parainfluenza virus detection Suspected infectious agents
Canine distemper virus RT-PCR Timing of specimen collection
Canine herpesvirus RT-PCR, antigen detection Type and amount of specimen to be collected
Feline herpesvirus-1 using IFA Transport conditions, including timing and method of
Feline calicivirus PCR transportation
PCR, IHC
RT-PCR, IHC

Eye coronavirus (FCoV), are difficult to isolate in cell culture or

Chlamydia felis PCR grow slowly, whereas others, such as feline calicivirus (FCV),
replicate readily and rapidly in cell culture, and the sensitivity of
Central Nervous System cell culture is high. Viruses differ in respect to the cell type they
Canine distemper virus RT-PCR, direct IFA, CSF prefer to replicate within. As a result, specimens should be sent
West Nile virus antibody detection to the laboratory with information on the specific viruses that
Encephalitis viruses RT-PCR are suspected.
RT-PCR The timing of specimen collection is particularly important
for viral infections. Specimens should be collected as early as
Gastrointestinal Tract possible following the onset of clinical signs, optimally within
Coronaviruses PCR the first week, because viral shedding may commence before
Canine distemper virus PCR, direct IFA/IHC the onset of signs and continue for only a few days. The dura-
Canine and feline parvovirus PCR, IHC tion of viral shedding depends on the type of virus and the
anatomic site sampled. When multiple animals are affected,
Genital collection of specimens from more than one animal may
Canine herpesvirus PCR increase the chance that an isolate will be obtained. If possible,
antibody testing using acute and convalescent phase serology
Congenital and Perinatal should be performed concurrently to help confirm the diagno-
Canine herpesvirus PCR sis (see Chapter 2).
Feline herpesvirus-1 PCR Selection of the best specimen and collection site for culture
is optimized based on knowledge of the pathogenesis of the
Blood infectious agent involved, because the optimum specimen col-
Feline leukemia virus PCR, antigen detection lection site may not be the site where clinical signs are most
Feline immunodeficiency virus PCR, antibody detection severe. Attempts should be made during specimen collection
Anaplasma phagocytophilum PCR, antibody detection to prevent contamination of the specimen with normal flora,
Rickettsia rickettsii PCR, direct IFA on skin although this is not always possible. Specimen size should also
Ehrlichia canis biopsies, antibody detec- be maximized (for example, at least 5 mL of blood, body flu-
tion ids, or lavage specimens, and ideally 8 to 10 mL of blood) to
PCR, antibody detection increase the chance of a positive isolation. In general, nasal or
nasopharyngeal washes have been preferred over nasal swabs
CSF, Cerebrospinal fluid; IFA, fluorescent antibody; IHC, immunohis- in human patients for isolation of respiratory viruses, but one
tochemistry; PCR, polymerase chain reaction; RT, reverse transcriptase.
study showed that nasal swab specimens were just as sensitive
as nasopharyngeal washes for isolation of most respiratory
transport and processing. Thus the actions of the veterinary cli- viruses.1 Nasal or oropharyngeal swab specimens are collected
nician play a critical role in ensuring positive test results when by placing a long-shafted swab in the area to be sampled, rotat-
a virus is present. ing the swab against the mucosa, and allowing the secretions to
The clinician should discuss with the laboratory what types be absorbed for approximately 5 to 10 seconds.
of viruses are suspected in light of the animal’s clinical presen- Swabs and small tissue specimens for virus isolation should
tation. The patient signalment, history, clinical signs, immune be placed in buffered virus transport medium, which contains
status, travel history, and number of animals affected should antibiotics and protein. This can be obtained from the labora-
be discussed to generate conclusions regarding the nature of tory or purchased from other commercial sources. It is impor-
the suspected infection (Box 1-1). Some viruses, such as feline tant that the medium used has not reached its expiry date.
4 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

TABLE 1-2
Specimen Collection Guide for Diagnosis of Viral and Intracellular Bacterial Infections of Companion Animals
System Affected Possible Agents Specimen Type
Respiratory Dogs: coronaviruses, canine adenovirus, Oropharyngeal swabs
tract influenza viruses, parainfluenza virus, Nasal flushes, transtracheal wash or bronchoalveolar lavage speci-
CDV, canine herpesvirus mens: ideally 5 to 10 mL of fluid
Cats: FHV-1, FCV, influenza viruses, Lung tissue obtained at biopsy or necropsy, including an area adja-
FCoV cent to affected tissue
Eye Dogs: canine herpesvirus, canine Conjunctival swab, scraping or biopsy
adenovirus
Cats: FHV-1, FCV, Chlamydia felis
Central nervous Dogs: CDV, West Nile virus, Cerebrospinal fluid: ideally at least 0.5 to 1 mL
system arboviruses Blood: 8 to 10 mL
Brain at necropsy
Gastrointestinal Dogs: CDV, CPV, rotaviruses, canine Feces: ideally an olive-sized portion of formed feces or 10 mL of
tract coronavirus liquid stool
Cats: FCoV, FCV, FeLV, rotaviruses, Intestinal biopsies obtained using endoscopy or surgery, or intesti-
toroviruses nal tissue obtained at necropsy
Genital Dogs: canine herpesvirus Vesicle scrapings, vaginal swabs
Cats: Chlamydia felis
Congenital and Dogs: canine herpesvirus Blood, tissues obtained at necropsy
perinatal Cats: FHV-1, FeLV
Blood Dogs: Anaplasma phagocytophilum, Blood: ideally 8 to 10 mL
Rickettsia rickettsii, Ehrlichia canis
Cats: FeLV, FIV, FCoV

CDV, Canine distemper virus; CPV, canine parvovirus; FCoV, feline coronavirus; FCV, feline calicivirus; FeLV, feline leukemia virus; FHV-1, feline
herpesvirus-1; FIV, feline immunodeficiency virus.

Liquid specimens such as blood, cerebrospinal fluid, and
bronchoalveolar lavage fluid do not need to be placed in trans-
port media. Blood samples should be collected using sterile tech-
nique, with antiseptic preparation of the site of venipuncture,
and can be submitted in EDTA anticoagulant tubes.
All specimens should be refrigerated on collection and trans-
ported as quickly as possible (preferably within 24 hours) to the
laboratory, because delayed transport can lead to loss of organ-
ism viability. If delays in excess of 2 to 3 days are anticipated,
the specimen can be frozen. Freezing should be avoided when-
ever possible, as it may lead to dramatic loss of virus viability.
If freezing is unavoidable, freezing at −70° C is preferable to
freezing at −20° C, and shipping on dry ice is preferable, if pos-
sible. The laboratory’s submission guide should be checked for
specimen handling recommendations.
Table 1-2 provides a guide to the recommended specimen
types for isolation of viruses or obligate intracellular bacteria
from companion animals. Specimens should be labeled with the
patient data, the site(s) from which the specimen(s) was collected,
specific organisms suspected, and the time and date of specimen
collection. Contained specimens should be placed inside leak-
proof triple packaging and transported on wet ice or cold packs
to the laboratory, especially if transport is expected to take lon-
ger than 1 hour. Absorbent materials should be placed within the
secondary container in order to absorb any spills. If specimens are
to be shipped, the specimen must be labeled and handled accord-
ing to governmental and International Air Transport Associa-
tion (IATA) regulations for shipping materials known to contain
infectious substances, which are categorized as Category A or
Category B. Category A infectious substances are those capa-
ble of causing permanent disability or life-threatening or fatal
disease in otherwise healthy animals and humans.2 Most speci-
mens submitted by veterinarians fall under Category B, which
are those that do not fall under the criteria for inclusion in Cat-
egory A. Updated documents providing guidance on regulations
for the transport of infectious substances are provided online by
the World Health Organization (WHO).2 Import permits may be
required for interstate and international transportation.
Diagnostic Methods
Maintenance of Cell Cultures in the Laboratory
In general, cells are grown as a monolayer on a plastic plate. The
cells in the monolayer can be derived directly from an animal
(primary cell culture), which tend to have a limited life span, or
they may be immortalized (continuous cell lines). Primary cell
cultures are needed for the isolation of some viruses, because
the cells more closely resemble those present in  vivo, and the
replication of these viruses occurs more efficiently in primary
cell lines than in continuous cell lines. Further subculture of pri-
mary cell lines often reduces their sensitivity to viral infection.
Primary cell cultures are generated by placing tissues in cell cul-
ture media, often after treatment of the tissue with an enzyme
such as trypsin or collagenase. Primary white blood cell cultures
(such as peripheral blood mononuclear cell cultures) are gen-
erated by separation of the white cells from the other cellular
elements using density gradient centrifugation, and adding them
to a culture medium. Ficoll, a highly branched polysaccharide,
 CHAPTER 1  Isolation in Cell Culture 5

TABLE 1-3
Examples of Continuous Cell Lines Used for Isolation of Viruses
and Intracellular Bacteria That Infect Dogs and Cats
Cell Line Cell Origin Pathogen(s)
Vero cells; African Green CDV11,12
recombinant monkey renal Rickettsia rickettsii13
Vero-SLAM epithelial cells Toxoplasma gondii14
cells
Madin-Darby Kidney CDV8,15
canine kidney Canine adenovirus8,15
cells (MDCK) Canine herpesvirus-18,15
Parvoviruses8,16
Canine parainfluenza A
virus8
Canine calicivirus4
Rotaviruses17
Influenza viruses18
FeLV19
Neospora caninum20
Crandell-Reese Fetal kidney FHV-121
feline kidney FCV21,22
cells FCoV23
Parvoviruses24
FIV25
HL-60 Human leukemia Anaplasma phagocyto-
philum26
A-72 Canine fibroma Canine adenovirus27
Canine coronavirus27
Canine parainfluenza B
virus27 FIGURE 1-1  A, Plastic flask that contains cell culture medium. B, A confluent cell
Canine herpesvirus27 monolayer is present on the bottom of the flask and can be visualized through the top of
McCoy Mouse fibroblast Chlamydia felis28 the flask using a binocular inverted microscope.
FCWF Felis catus whole FCoV29
different viruses prefer to replicate in differing cell types. Mixed
fetus, has FHV-130
cell cultures are also now available commercially to simultane-
­characteristics
ously facilitate isolation of multiple different viral pathogens.
of macrophages
Cells for cell culture are stored in the laboratory in liquid
DH-82 Monocyte/macro- Ehrlichia canis31 nitrogen tanks. The cells are thawed, dispersed in cell culture
phage medium, and allowed to settle on the bottom of a plastic flask
(Figure 1-1). The cell culture medium keeps the cells moist and
CDV, Canine distemper virus; FCoV, feline coronavirus; FCV, feline
calicivirus; FeLV, feline leukemia virus; FHV-1, feline herpesvirus-1; provides the cells with nutrients. Minimum essential medium
FIV, feline immunodeficiency virus. (MEM), also known as Eagle’s minimum essential medium, and
Dulbecco’s medium are examples of widely used synthetic cell
culture media. The cell culture medium contains a balanced salt
is an example of a medium used commonly for density gradient solution, essential amino acids, glucose, vitamins, and a bicar-
centrifugation. Primary cell cultures have been used widely for bonate buffering system. Variations of MEM are available, some
the isolation of intracellular pathogens of dogs and cats.3-6 of which contain nonessential amino acids, the pH indicator phe-
Low-passage cell lines remain viable and sensitive to viral nol red, and the pH buffering agent HEPES (4-(2-hydroxyethyl)-
infections for 20 to 50 passages. Continuous cell lines are the type 1-piperazineethanesulfonic acid), which helps to maintain a
of cell line used most commonly for diagnostic, research, and physiologic pH despite an increasing concentration of CO2 that
commercial purposes. These are derived from cancer cells (such as is produced as a result of cellular respiration. The medium often
the widely used HeLa cell line, derived from human cervical can- requires supplementation with animal serum, most commonly
cer cells of a patient named Henrietta Lacks),7 or they result from fetal calf serum, which helps the cells to attach to and spread
experimental induction of cellular mutations (for example, using on the plate, although serum-free medium has also been used
a carcinogen). Continuous cell lines representing a wide variety successfully for culture of canine and feline viruses.8 Media for
of cell types are available from commercial suppliers (Table 1-3). cell culture growth differ from media for maintenance of cells in
Laboratories that perform virus isolation for disease diagnosis culture, the former generally containing a higher concentration
may need to simultaneously inoculate multiple cell lines, because of animal serum (10% versus 2% for maintenance medium).
6 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
To prevent bacterial or fungal contamination when viral
pathogens are cultured, careful attention to sterile technique is
required. Manipulation of cells in the flask is performed in a
laminar flow hood, and the culture medium is sometimes sup-
plemented with antibiotics (generally ampicillin or an amino-
glycoside), with or without an antifungal agent (amphotericin B
or nystatin). Cell culture flasks are placed in a humidified incu-
bator that maintains temperature usually at 37°C, and a gas
mixture that typically contains 5% CO2.
The cell culture flasks are removed from the incubator and
examined daily using an inverted binocular tissue culture micro-
scope (see Figure 1-1), which allows examination of unstained
cells through the bottom of the flask. The medium is replaced
with fresh medium if the pH indicator suggests it is becoming
too acidic. Once the cells have multiplied sufficiently to form a
semiconfluent monolayer, the cells can either be passaged or be
allowed to reach confluence before inoculation with specimen.
In order to passage the cells, the medium is removed, and a small
volume of trypsin or EDTA solution is added to the monolayer.
The monolayer is incubated with the solution for several min-
utes, after which the cells detach from the flask and can be resus-
pended in medium, which is then added to new flasks.
Occasionally, cell lines become cross-contaminated with other
cell lines. The HeLa cell line is an example of a prolific and hardy
cell line that commonly contaminates other cultures. Major cell
line repositories such as the American Type Culture Collection
(ATCC) use genetic typing methods to verify the identity of the
cells in their collection. Cell lines can also become easily con-
taminated with Mycoplasma spp., which pass through filters
used to exclude other bacteria. The presence of contaminating
Mycoplasma spp. can interfere with replication of other patho-
gens and cause alterations of cellular morphology. Mycoplasma
contamination is detected using stains such as Hoechst stain,
culture for Mycoplasma, and commercially available PCR assays
that specifically detect Mycoplasma DNA for the purpose of cell
culture quality assurance.9
A B
FIGURE 1-2  A, Cytopathic effects produced by a herpesvirus as viewed in the laboratory. Note the focal areas of rounded and detached cells. B, Hemadsorption: erythrocytes adsorb
to infected cells that have incorporated hemagglutinin into the plasma membrane. Magnification ×60. (Courtesy of Jack I; from MacLachlan NJ, Dubovi EJ eds. Fenner’s veterinary virology,
4 ed. New York: Academic Press, 2011.)
Inoculation of Cell Cultures
Infection of a cell monolayer is accomplished by removal of
overlying medium and inoculation of the monolayer with a sus-
pension of viral or bacterial organisms. If tissue specimens are
provided for culture, the specimens are homogenized in culture
medium before they are inoculated onto the monolayer. Speci-
mens may first be centrifuged to remove cell debris and bacte-
ria. Passage through a filter can also be used before inoculation
to remove bacteria. The inoculum is added to the monolayer.
Organisms within the inoculum are then allowed to settle on
the monolayer for approximately 1 hour before the residual
inoculum is removed and fresh maintenance medium is added.
For isolation of virus, the monolayer is then examined daily
for evidence of cytopathic effect (CPE). Medium is replaced
weekly or biweekly. CPE refers to the cellular changes induced
by viral replication in the cell culture monolayer and includes
cell lysis or cell fusion (syncytium formation). The presence of a
particular viral pathogen is indicated by the appearance of char-
acteristic CPE for that pathogen after a predicted incubation
period (Figure 1-2). For example, CPE induced by FCV is char-
acterized by cell rounding and detachment that can occur within
24 hours, and within 16 hours for highly virulent strains.10
The presence of CPE is confirmed through the comparison of
inoculated cultures to uninoculated (control) cultures. Exami-
nation of the cell monolayer using light microscopy after fixa-
tion and staining can reveal additional diagnostic features, such
as the presence of inclusion bodies and syncytium formation.
Some viruses are relatively noncytopathogenic. In some cases,
the presence of these viruses can be demonstrated by adding
washed erythrocytes to the monolayer and examination of
the monolayer for evidence of hemadsorption (see Figure 1-2,
B). Hemadsorption results from incorporation of viral surface
hemagglutinin molecules into the plasma membranes of the cell
culture monolayer. Examples of hemadsorbing viruses that pro-
duce minimal CPE include influenza and parainfluenza viruses.
Other, more sensitive and specific methods for identification of

FIGURE 1-3  Indirect fluorescent antibody detection of noncytopathic virus infected
cells. A cell monolayer exposed to virus for 72 hours was fixed with cold acetone. Fixed
cells were stained with a mouse monoclonal antibody specific for bovine viral diarrhea
virus (BVDV) followed by a goat anti-mouse serum tagged with fluorescein isothiocyanate.
(From Maclachlan NJ, Dubovi EJ. Fenner’s Veterinary Virology, 4 ed. New York: Academic
Press, 2011.)
FIGURE 1-4  Schematic of the shell vial virus isolation technique. (Modified from
Shelhamer JH, Gill VJ, Quinn TC, et al. The laboratory evaluation of opportunistic pulmo-
nary infections. Ann Intern Med 1996;124(6):585-599.)
an organism that has infected a cell culture monolayer include
fixation and staining of the monolayer with fluorescent anti-
bodies that are specific for certain viruses (Figure 1-3), use of
nucleic acid detection techniques such as PCR, or use of electron
microscopy.
For spin-amplified cell culture, the monolayer is centrifuged
at low speed after inoculation. This can enhance recovery of
certain viruses and intracellular bacterial pathogens such as
chlamydiae. Either an entire plate of cell culture wells can be
spun in a plate centrifuge, or shell vials can be used (also known
as the shell vial technique). Shell vials are small, flat-bottomed
bottles (Figure 1-4). A monolayer is grown on a glass coverslip
at the bottom of the vial, and after inoculation, the vials are
centrifuged at low speed. Using this method, fluorescent anti-
body staining for viral or chlamydial antigen is used to detect
the pathogen before CPE occurs. After 48 to 72 hours, the cov-
erslips are fixed, stained with virus-specific fluorescent-labeled
CHAPTER 1  Isolation in Cell Culture 7
A B C
D E F
FIGURE 1-5  Determination of the concentration of infectious virus using a plaque
assay. Vero cell monolayers were inoculated with serial 10-fold dilutions of vesicular sto-
matitis virus. After a 1-hour adsorption period, cultures were overlayed with 0.75% aga-
rose in cell culture medium containing 5% fetal bovine serum. Cultures were incubated for
3 days at 37°C in a 5% humidified CO2 atmosphere. The agarose overlay was removed and
cultures fixed and stained with 0.75% crystal violet in 10% buffered formalin. A, Control
culture. B-F, serial 10-fold dilution of virus: B, 10−3; C, 10−4; D, 10−5; E, 10−6; F, 10−7.
(From Maclachlan NJ, Dubovi EJ. Fenner’s Veterinary Virology, 4 ed. New York: Academic
Press, 2011.)
antibodies, removed from the vials, and examined with a fluo-
rescence microscope.
Plaque Assays
Overlay of the monolayer with agar or methylcellulose after
inoculation with virus minimizes subsequent viral movement
through the monolayer and restricts damage by each replicating
viral particle to a small area. This results in the formation of
“holes” in the monolayer, or plaques (Figure 1-5). The mono-
layer can then be stained and the number of plaques can be
counted in order to obtain information regarding the amount
of virus in the inoculum. Plaque assays are commonly used by
researchers to assess the efficacy of antiviral treatments, through
reduction of plaque formation.
Laboratory Safety Concerns That Relate
to Cell Culture
The processing of specimens in a biological safety cabinet not
only serves to protect cultures from contamination but also
acts to protect the laboratory worker from ­laboratory-acquired
infections. Most viruses grown in veterinary diagnostic labora-
tories are classified as biosafety level 2 (BSL 2) agents. Biosafety
levels range from 1 through 4 (Table 1-4). In the United States,
the Centers for Disease Control and Prevention (CDC) assigns
these levels. It is important that veterinary clinicians be aware
that isolation of hazardous pathogens can only be conducted in
specially designed and accredited laboratories.
Interpretation of Results
Negative Results
Reasons for negative test results using virus isolation are numer-
ous, so the clinician should not assume that a virus is not pres-
ent when a negative test result is obtained (Box 1-2). Negative
test results can occur as a result of inadequate specimen size,
lack of viral shedding by the animal at the time of specimen col-
lection, the binding of antibodies to viruses within the specimen,
8 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

TABLE 1-4
Biosafety Levels Set for Diagnosis of Laboratory Practices
Level Risk Examples Example Precautions
BSL 1 Minimal potential hazard to human Canine adenovirus-1 Gloves, facial protection. Standard microbiologic
health and the environment Nonpathogenic practices using bench-top techniques. Routine
Escherichia coli decontamination practices (hand washing, routine
bench disinfection, autoclaving of infectious waste).
BSL 2 Moderate potential hazard to Most veterinary Access to the laboratory restricted when work is taking
human health and the environment. viruses, including place; extreme precautions with sharp contaminated
Organisms cause mild disease or are influenza viruses materials; use of appropriate biosafety cabinets
difficult to contract as laboratory when generation of aerosols possible. No require-
aerosols. ment for directional airflow into the laboratory.
BSL 3 Dangerous agents that can be West Nile virus Laboratory is located away from high-traffic areas.
transmitted by aerosol within the Equine encephalitis Restricted laboratory access when work in progress;
laboratory but for which effective viruses double door entry, ventilation providing airflow
vaccines or treatments exist. Rickettsia rickettsii into the room, exhaust air not recirculated; special
Coxiella burnetii practices and protective clothing for BSL 3, including
Mycobacterium tuber- biosafety cabinet use; special floor and ceiling
culosis materials specified.
BSL 4 Dangerous and exotic agents that pose Ebola virus Hazmat suit and self-contained oxygen system, entrance
a high risk of aerosol-transmitted Marburg virus containing multiple showers, a vacuum room, an
laboratory infections, or which Smallpox ultraviolet light room, and multiple airlocked doors.
produce severe or fatal disease in Strict control of laboratory access to authorized
humans personnel.

BOX 1-2
Reasons for Negative Test Results following Isolation
of Viruses in Cell Culture
Virus not causing the disease
Virus causing the disease, but:
Specimen size inadequate
No viral shedding at the time of sampling
No viral shedding at the site of sampling
Antibody interference with viral infectivity for cell
culture
Inadequate organism numbers at the anatomic site
of specimen collection
Loss of organism viability during transportation to
the laboratory
Overgrowth of one viral pathogen by another
Overgrowth by bacterial or fungal agents
Lack of cell sensitivity for the virus (wrong cell type
inoculated)
Laboratory inexperience with techniques required
for virus isolation and identification
inadequate organism numbers at the anatomic site of specimen
collection, or loss of organism viability during transportation
to the laboratory. Loss of viral viability is more likely to occur
with enveloped viruses such as canine distemper virus than with
non-enveloped, hardy viruses such as canine parvovirus or FCV.
Negative test results can also occur when the cell line inocu-
lated is not sensitive to the virus present in the specimen. Low-­
passage cells may also lose their infectivity for viral infection if
they have undergone multiple serial passages.
False negatives can also occur if the plates are overgrown by
bacteria or fungi. Although treatment of the viral transport and
culture media with antimicrobials can help prevent this, resis-
tant bacteria or fungi may still be present. Sometimes, multiple
viruses are present within a specimen, and one virus overgrows
another. For example, this can occur with mixed infections with
FCV and feline herpesvirus-1 (FHV-1) in cats with upper respi-
ratory tract disease. FCV rapidly infects Crandell-Reese feline
kidney cells and produces CPE, which obscures the concurrent
presence of FHV-1.
Positive Results
It is imperative that veterinary clinicians be aware that the
detection of a virus in a specimen does not always imply that
the organism is the cause of the animal’s clinical signs. This is
especially true for specimens collected from the respiratory or
gastrointestinal tracts, where multiple infectious agents may
be present concurrently, and in many cases, viruses can repli-
cate in these locations without causing clinical signs of illness.
In some animals, the development of severe clinical signs is
more likely when there is simultaneous presence of multiple
infectious agents. As noted previously for specimens that test
negative, the presence of one agent may also obscure the pres-
ence of another, more significant organism (such as with FCV
and FHV-1 co-infections), which could result in the incorrect
assumption that only one organism is the cause of an animal’s
disease.

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CHAPTER 2
Immunoassays
Jane E. Sykes and Shelley C. Rankin
w KEY POINTS
• I mmunoassays are commonly used in practice to detect anti-
gen or antibody in body fluids.
• The term serology is most often used to refer to assays that
detect antibody in serum.
• Immunoassays that detect antigen or antibody in dogs and cats
suspected to have infectious diseases include direct and indi-
rect immunofluorescent antibody (IFA), immunohistochemistry
(IHC), ELISA, Western immunoblotting, agglutination tests, gel
immunodiffusion assays, and serum neutralization.
• Positive immunoassay results do not always imply infection,
and negative results do not rule out infection.
INTRODUCTION
Serology refers to the measurement of antigen-antibody interac-
tions for diagnostic purposes, but is most often used to refer to
the detection of antibodies in serum. In general, a blood sample
is seroreactive (or seropositive) if it contains antibodies to a par-
ticular pathogen. Assays have been designed that detect differ-
ent classes of antibodies in serum, most commonly IgG or IgM.
Methods used to detect antibodies can also be applied to detect
antigen. They can also be used on body fluids other than serum,
such as urine, cerebrospinal fluid, and aqueous humor. Assays that
measure antigen-antibody interactions for diagnostic purposes
are most broadly referred to as immunoassays. Types of immu-
noassays include immunofluorescent antibody (IFA) tests, ELISAs,
agglutination tests, gel immunodiffusion tests (AGID or gel ID
tests), and Western blotting. Many of these assays involve the use
of polyclonal antibodies, or, more commonly, monoclonal antibod-
ies, which are commercially available. Polyclonal antibodies are
produced in animals (e.g., rabbits or horses) as a normal antibody
response to antigen exposure. Monoclonal antibodies are pro-
duced in almost unlimited amounts in tissue culture, and have a
higher degree of antigenic specificity.
Some immunoassays, such as ELISA and IFA, provide quantita-
tive results, expressed as optical density units (ODs) or titers (e.g.,
1:800, or reciprocal titer = 800). Exposure to an infectious agent
results in a rise in antibody titer over time (Figure 2-1). Usually, the
first antibody class to be produced by plasma cells is IgM, after
which IgG synthesis by plasma cells occurs. When exposure to a
pathogen occurs for the first time, there is a lag phase of 5 to 7 days
before IgM antibodies can be detected in the blood (primary anti-
body response). The antibody titer increases, and if the infectious
agent is cleared, there is a progressive decline in the titer over weeks
to months. The speed and magnitude by which the antibody titer
rises depends on host factors and the infectious agent involved. For
10
• W  hen interpreting the results of immunoassays, veterinarians
should consider what is being measured (antigen or antibody),
the likelihood of antigen or antibody being present in relation
to the onset of illness, whether the positive test result is consis-
tent with the animal’s clinical signs, and the animal’s vaccina-
tion history and immune status. The possibility of false positives
due to immunologic cross-reactivity, as well as the analytical
sensitivity and specificity of the test used (i.e., the test’s perfor-
mance), must also be considered.
• Additional assays may be required to support immunoassay
test results.
some diseases, such as leptospirosis, a significant rise occurs within
3 to 4 days of the onset of clinical signs. In secondary, or anamnes-
tic, immune responses, the lag phase is shorter, the antibody titer
increases more rapidly and to a greater magnitude, and antibodies
may persist for longer periods of time (months to years).
Serologic diagnosis of infectious diseases relies on an understand-
ing of the timing of the antibody response in relation to development
of clinical signs for different infectious diseases. The results of sero-
logic testing are interpreted differently for acute infections than
they are for chronic or persistent infections. For infections with a
short incubation period, such as leptospirosis or Rocky Mountain
spotted fever, antibody titers are often negative in the first week of
illness. In general, an increase in antibody titers over a 2- to 4-week
period then occurs. Seroconversion is consistent with recent infec-
tion, and at least a fourfold increase in titer is required for the
change to be considered significant. Because serologic test results
can differ between laboratories that perform the same assay, the
same laboratory should be used to determine the acute and the
convalescent titer. Ideally, to minimize interassay variation, an ali-
quot of the acute specimen should be stored, frozen, and assayed
at the same time as the convalescent specimen, although in reality
this is often not done. A fourfold decline in antibody titer may also
be consistent with recent infection, depending on the stage of ill-
ness at which serologic testing is performed. For chronic, persistent
infections, such as FIV infection or canine leishmaniosis, a single,
positive antibody titer can indicate active infection. Titer increases
do not occur when infection persists beyond 1 to 2 months, and so
acute- and convalescent-phase serology is not useful for diagnosis
of chronic infections.
Specimen Collection and Transport
Blood collected for antibody testing should be allowed to clot,
and the serum separated immediately and then refrigerated or
 CHAPTER 2  Immunoassays 11

FIGURE 2-1  Time course of primary and secondary immune responses, showing rela- FIGURE 2-2  Direct fluorescent antibody stain of brain tissue for rabies virus.
tive amounts of IgM and IgG produced at different times after antigen exposure. (Modified Frozen tissue sections were stained with a commercial reagent that contains three mono-
from Tizard IR. Veterinary Immunology, 8 ed. St Louis: Saunders; 2008) clonal antibodies specific for the rabies virus nucleocapsid. Antibodies were labeled with
fluorescein. Positive staining is present in a Purkinje cell. (Courtesy of Galligan J. New York
State Department of Health. In Maclachlan NJ, Dubovi EJ. Fenner’s Veterinary Virology, 4
frozen. Specimens can be refrigerated for up to a week before ed. New York: Elsevier; 2011.)
they are shipped to the laboratory. If testing is to be delayed for
longer than a week, specimens should be frozen at −20°C or
−80°C. Specimens can be stored frozen for years without loss of
antibody titer. For specimens collected for antigen testing, assay
results may be more susceptible to variation with specimen stor-
age. The laboratory should be contacted in order to determine
specimen storage and handling requirements.

Diagnostic Methods
Immunofluorescent and Immunoperoxidase
Antibody Assays
Direct IFA (also known as direct FA) detects antigen in clinical
specimens. IFA involves the use of an antibody that has been
tagged (conjugated) with a fluorescent label (such as fluores-
cein isothiocyanate) to detect the presence a specific antigen.
IFA is performed directly on a glass slide, and fluorescence is A
detected by microscopy. The sensitivity of direct IFA is too
low to detect individual virus particles or soluble antigen, so
usually IFA detects antigen present in association with eukary-
otic or bacterial cells. If the antigen is intracellular, pretreat-
ment of the slide to permeabilize the cells may be necessary.
The glass slide is then incubated with a solution that contains
the fluorescent antibody, then washed to remove unbound
antibody, and the cells are examined using a fluorescence
microscope (Figure 2-2). Examples of the use of direct IFA
in veterinary medicine include detection of Giardia oocysts,
FeLV within monocytes in peripheral blood or bone marrow,
or canine distemper virus within epithelial cells from a con-
junctival scraping. Nonspecific fluorescence can result in false
positives using these methods, but this can be overcome with
the inclusion of proper controls and technical expertise. An B
alternative to the use of a fluorescein-conjugated antibody is
the use of an antibody that has been conjugated to an enzyme FIGURE 2-3  Immunohistochemistry. A, Hematoxylin and eosin stained section of a
such as horseradish peroxidase (immunoperoxidase). A sub- lingual ulcer from a cat infected with feline herpesvirus-1. B, The brown stained cells lining
the ulcer are positive using an immunohistochemical stain for feline herpesvirus-1. (Cour-
strate, most commonly 3,3′-diaminobenzidine (DAB), is then
tesy of Dr. Patricia Pesavento, School of Veterinary Medicine, University of California, Davis.)
added to the slides. In the presence of hydrogen peroxide, DAB
is converted to an insoluble brown precipitate, which can be Indirect IFA (also known as IFA testing, or IFAT) is generally
visualized using conventional light microscopy. This technique used to establish a serum antibody titer to an infectious agent,
is known as immunocytochemistry. The same method, when but it can also be used to detect antigen. When used to detect
applied to tissue sections, is known as immunohistochemistry antigen, it is more sensitive than direct IFA. For measurement of
(IHC) (Figure 2-3). antibody titers, patient serum is serially diluted in the laboratory
12 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
FIGURE 2-4  Flow-through immunoassay device (IDEXX Laboratories, Inc.). Specific antigen (for example, a recombinant protein) or antibody is immobilized on a membrane in the
device in a spot or line. 1, Patient serum that may or may not contain target antigen or antibody is mixed with a solution that contains conjugated antibody or antigen. 2-3, The solution
is allowed to flow through the device, where it becomes captured on the membrane. 4, The device is activated (snapped), which releases wash and substrate reagents within the device.
A positive reaction appears as a colored bar, or in this case, a dot. Color development in designated spots on the membrane can indicate the presence of antibody to Anaplasma spp., Ehrlichia
canis, and Borrelia burgdorferi, or the antigen of Dirofilaria immitis.
and reacted with a known antigen that has been fixed to glass
slides. IFA slides are commercially manufactured for this pur-
pose. Slides are then washed to remove unbound antibody. A
secondary antibody, which is designed to react with the bound
patient antibodies (e.g., dog IgG or dog IgM), is applied to the
slides, which are then washed again. This secondary antibody
has been conjugated to a fluorescent label. The slides are then
examined using a fluorescence microscope. The highest dilution
of serum that results in specific fluorescence is reported to the
clinician as the antibody titer to the infectious agent of interest.
Examples of the use of indirect IFA for serologic testing in dogs
and cats include quantitative serology for some tick-borne infec-
tious diseases (e.g., Ehrlichia canis, Anaplasma spp.).
More recently, veterinary immunofluorescence assays have
been marketed that allow multiple antigen (or antibody) targets
to be mounted on novel platforms, such as fluorescent beads
or wells on the surface of a spinning silicon disc that resem-
bles a compact disc. Automated silicon disc–based assays allow
rapid and simultaneous analysis of hundreds of specimens, with
dramatic reduction in labor associated with traditional IFA or
ELISA assays, and reduced chance of false-positive or -negative
assay results due to human error. A bead-based assay is cur-
rently available in the United States for detection of antibody
to Borrelia burgdorferi (see Chapter 51). A silicon disc–based
assay is available in the United States for detection of antibodies
to B. burgdorferi, E. canis, and Anaplasma phagocytophilum
and antigen to Dirofilaria immitis (Accuplex 4, Antech Diag-
nostics, Irvine, Calif). The performance of these assays in the
field is under investigation.
Enzyme Linked Immunosorbent Assays
ELISA (also known as enzyme immunoassay) can be used to
detect either antigen or antibody. In the laboratory, it is usually
performed in a polystyrene microtiter plate. However, in-clinic
modifications of the test, such as flow-through or lateral-flow
ELISAs (e.g., SNAP, IDEXX Laboratories, Inc.), whereby anti-
gen or capture antibody is immobilized on a membrane filter, are
widely used in veterinary practices (e.g., for diagnosis of feline
retroviral, heartworm, Giardia, Leishmania, and tick-borne
infections) (Figure 2-4). For antigen detection, an unknown
quantity of antigen in a clinical specimen is immobilized in the
 
wells of the plate. The antigen is either bound directly to the
surface of the plate or is bound through the use of a capture
antibody, which is an antibody that is bound to the plate sur-
face that specifically binds to the antigen of interest (sandwich
ELISA). The antigen is then detected with an antibody that is
conjugated to an enzyme such as immunoperoxidase, and the
substrate is added. This results in a color change, which can be
read visually or using an ELISA plate reader (Figure 2-5). The
plate reader records optical density of the wells in comparison
to that of control wells, allowing assessment of the quantity of
antigen present. The plates are washed with a detergent solution
between steps, and this can be automated using an ELISA plate
washer. Sandwich ELISAs have been used widely for detection
of FeLV antigen in cat blood. In competitive ELISA, the test
specimen (e.g., dog serum), which contains an unknown amount
of antigen, is mixed with a known amount of labeled antigen.
The mixture is added to wells that contain known amounts of a
capture antibody, and the labeled and unlabeled antigens com-
pete for antibody binding. The more labeled antigen that binds,
the less antigen is present in the test specimen. Other variations
of competitive ELISAs also exist.
For antibody detection, serial dilutions of patient serum can
be added to a known amount of antigen fixed on a plate and
detected using a secondary antibody.
Western Immunoblotting
Western immunoblotting, also known as Western blotting, is
also used to detect antibodies to an infectious agent in patient
serum. After physical and/or chemical disruption of an organ-
ism, the proteins are then separated on the basis of molecular
mass using vertical sodium dodecyl sulfate–polyacrylamide gel
electrophoresis (SDS-PAGE). The protein bands are then trans-
ferred (blotted) to a nitrocellulose membrane using an electric
current. The membrane is cut into vertical strips, and each
strip is probed with serial dilutions of patient serum. If there
are antibodies in the serum to antigens from the organism of
interest, the antibodies bind to the antigens on the strip. The
antigen-antibody complexes can then be visualized as discrete
bands using immunoperoxidase detection methods. The pattern
of band reactivity is specific for an immune response to that
organism (Figure 2-6). Western blotting is offered commercially
 CHAPTER 2  Immunoassays 13

FIGURE 2-5  Sandwich ELISA. An antibody is immobilized in the plate, which binds antigen in a test specimen. The antigen is then detected using an antibody that is conjugated to an
enzyme such as immunoperoxidase, and the substrate is added, which results in a color change. (Modified from Tizard IR. Veterinary Immunology, 8 ed. St Louis: Saunders; 2008.)

FIGURE 2-6  Western blotting (immunoblotting). Purified viruses or bacteria are disrupted and subjected to electrophoresis along the length of a polyacrylamide gel, so that the
proteins are separated according to their molecular weight (A). The bands are then transferred to a nitrocellulose paper membrane (B) and probed with patient serum (C). The strip shown
on the right is a Western blot that shows antibody reactivity to the antigens of FIV (D). (Original blot courtesy of IDEXX Laboratories, Inc.)

for the diagnosis of FIV and B. burgdorferi infection and has
been used in research laboratories as the gold standard for the
detection of antibody responses to vector-borne pathogens such
as E. canis. In the case of FIV infection, Western blotting has
been used to confirm positive ELISA test results.1 In the case of
B. burgdorferi infection, Western blotting has been evaluated
for its ability to differentiate between the response to vaccina-
tion and natural exposure.2
Agglutination Testing
Agglutination tests detect antibody or antigen and involve agglu-
tination of bacteria, red cells, or antigen- or antibody-coated
latex particles. They rely on the bivalent nature of antibodies,
which can cross-link particulate antigens. Serial dilutions of
serum are tested for their ability to cause or inhibit agglutination,
and the highest dilution that causes or inhibits agglutination is
reported as the antibody or antigen titer. IgM causes aggluti-
nation more effectively than IgG. If excess antibody is present,
particles may become so coated with antibody that agglutina-
tion is actually inhibited. This is called the prozone effect and
can result in false-negative test results if serum is not adequately
diluted. Examples of agglutination tests that are used to diag-
nose infectious diseases of dogs and cats include the microscopic
agglutination test (MAT) for serologic diagnosis of leptospirosis
14 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
A for cytopathic effect or immunostaining) is the antibody titer
B antibodies are present in the serum, the addition of this antigen
FIGURE 2-7  A, Gel immunodiffusion (or double diffusion) system used for detection of
antibodies to Aspergillus fumigatus, Histoplasma capsulatum, Blastomyces dermatitidis, and
Coccidioides immitis (Meridian Bioscience, Inc.). The kit consists of an agar diffusion medium
in a small plastic tray that contains four arrays of six wells arranged around a central well.  sheep RBCs. A lack of hemolysis indicates a positive CF test.
An antigen preparation for each of the fungal species is placed in the central well of each
array. B, Positive gel immunodiffusion for Aspergillus fumigatus. The central well (Ag)
contains a purified carbohydrate preparation from mycelial phase cultures of Aspergillus
fumigatus, Aspergillus niger, and Aspergillus flavus. The top two wells (D1) and the bottom
two wells (D2) contain serum from 2 dogs (dog 1 and dog 2) with nasal aspergillosis (per-
formed in duplicate for each dog). After the wells are loaded, the plate is incubated at room
temperature for 24 hours. Precipitin lines form when the antigen and antibody diffuse out
of each well and form immune complexes. A light box is used to illuminate the plate so the
precipitin bands are visible. Both dogs are positive for antibody to the Aspergillus species
tested (small arrows), although the reaction is strongest for dog 1. The arrowheads are the
precipitin lines generated by the positive control antibodies (in wells labeled C).
(agglutination of live leptospires) and the cryptococcal antigen
latex agglutination test (agglutination of antibody-coated latex
beads). Hemagglutination inhibition (HI) is used to determine
antibody titers to canine parvovirus and canine influenza virus,
and it evaluates the ability of serum to inhibit erythrocyte agglu-
tination by these viruses.
Agar Gel Immunodiffusion
For agar gel immunodiffusion (AGID, gel ID or double diffu-
sion) tests, round wells are cut in a layer of agar on a plastic plate
using a punch. One well, usually the central well, is filled with a
soluble antigen, and the other wells are filled with patient serum.
A positive antibody control specimen is included in each assay.
The reagents are allowed to diffuse into the agar, and when anti-
bodies and antigen meet in optimal proportions (the point of
equivalence), immune complexes form. This results in a visible
line of precipitate between the antigen well and wells that contain
positive serum (Figure 2-7). Immunodiffusion testing is most often
used to detect antibodies to fungal pathogens in dogs, such as
Aspergillus fumigatus,3 Coccidioides immitis,4 and Blastomyces
dermatitidis.5 It is also used to detect antibodies to Brucella canis.
Serum Neutralization
Serum neutralization is a technique used to determine the anti-
body titer to viruses. Serial dilutions of patient serum are mixed
with a known quantity of virus particles. The mixture is incu-
bated to allow the antibodies in the patient serum to bind to the
virus particles, and the mixture is then added to a confluent cell
monolayer. The highest dilution of patient serum that inhibits
viral replication in the monolayer (as determined by examination
reported by the laboratory. Serum neutralization can be used to
detect antibodies to canine distemper virus and several respira-
tory viruses that infect dogs and cats.
Complement Fixation Testing
Complement fixation (CF) tests have largely been superseded by
the use of ELISA tests. The CF test detects antibodies in patient
serum. The serum is heat-treated to inactivate complement in the
test specimen. The test uses sheep RBCs, anti–sheep RBC anti-
body, and unheated guinea pig serum as a source of complement.
Under normal circumstances, when these three reagents are mixed,
the result is hemolysis. Hemolysis occurs when complement binds
to antibody-coated RBC and triggers formation of the membrane
attack complex, with resultant pore formation. In the CF test, a
known antigen is added to the heat-treated patient serum. When
leads to the formation of antigen-antibody complexes. Guinea pig
complement is then added to the mixture and is consumed by the
complexes. The resultant mixture is thus unable to lyse sensitized
Interpretation of Results
To interpret the results of serology or immunoassay tests, the
clinician first needs to consider whether the test was designed to
detect antibody or antigen (Tables 2-1 and 2-2), and, if designed
to detect antibody, whether it was IgM-specific (detects early
antibody responses). The results also have to be interpreted in
light of the pathogenesis of the infectious disease, specifically
the timing of antigen and antibody appearance in the body fluid
being tested. Finally, the sensitivity and specificity of the test
itself need to be considered (i.e., the analytical sensitivity and
specificity), which are determined during test validation (Table
2-3). For example, some immunoassays that detect antigen will
not generate a positive result unless a large amount of antigen
is present in the specimen provided (low analytical sensitivity).
Test analytical sensitivity and specificity should be available
from the laboratory that performs the test. False-positive test

TABLE 2-1
Guidelines for Interpretation of Test Results Using Immunoassays That Detect Antigen
Antigen Test Result Possible Explanation
Negative Pathogen of interest not present
Antigen present, but quantity below
limit of detection
Antigen present but complexed by
antibody and not available for
detection
Technical error or reagent failure
Positive Pathogen of interest present
Antigen present but viable microbe
absent
Vaccine antigen present
Cross-reactive antigen present
from another substance or
pathogen
Technical error
Poor assay analytical specificity
(prone to generate false-positives)
TABLE 2-2
Guidelines for Interpretation of Test Results Using Immunoassays That Detect Antibody
Antibody Test Result Possible Explanation
Negative Exposure to pathogen of interest
did not occur
Too early in course of infection/
illness
Severe immunosuppression
Technical error
Poor assay analytical sensitivity
(prone to false-negatives)
Positive Previous natural exposure to the
pathogen of interest
Previous immunization against
the pathogen of interest
Immunologic cross-reactivity
Technical error
Poor assay specificity (prone to
­false-positives)
CHAPTER 2  Immunoassays 15
Troubleshooting Suggestions
Confirm with additional diagnostic test if necessary, (e.g.,
alternative antigen assay, antibody or PCR assay)
Consider an antibody or PCR assay
Consider an antibody or PCR assay
Identified with inclusion of appropriate laboratory controls
Confirm with additional diagnostic test if necessary, especially
in regions with a low prevalence of infection. Antigen pres-
ence may not imply that the pathogen is the cause of disease
Confirm with additional diagnostic test
Obtain vaccination history and consider a test that discrimi-
nates between vaccine and wild-type organisms, if available
Obtain history and consider the likelihood of infection with
related agents
Identified with inclusion of appropriate controls by the
­laboratory
Use alternative assay
Troubleshooting Suggestions
Confirm with additional diagnostic test if necessary
Obtain convalescent titer; consider an antigen or PCR test for
early diagnosis
Consider an antigen or PCR test
Identified with inclusion of appropriate controls by the
­laboratory
Use alternative assay
If infection is chronic and persistent, positive test results may
equal active infection.
Obtain vaccination history; consider use of assays that dis-
criminate between natural exposure and vaccine antibody, if
available
Obtain history and consider the likelihood of previous exposure
to related agents
Identified with inclusion of appropriate controls by the
­laboratory
Use alternative assay. Consider the use of a confirmatory test,
especially in regions with low disease prevalence
16 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
TABLE 2-3
Sensitivity, Specificity, Positive and Negative Predictive Value
Test Attribute Definition
Sensitivity Number of truly positive animals that are
identified as positive by the test, divided
by the total number of animals that are
truly positive (as determined by the gold
standard assay) (%). A test with low
sensitivity fails to detect many positive
animals (many false-negative results).
Specificity Number of truly negative animals that are
identified as negative by the test, divided by
the total number of animals that are truly
negative (as determined by the gold stan-
dard assay) (%). A test with low specificity
generates many false-positive test results.
Positive pre­ The number of animals that are truly
dictive value positive divided by the total number of
animals that test positive (true positives
plus false positives).
Negative pre­ The number of animals that are truly
dictive value negative divided by the total number of
animals that test negative (true negatives
plus false negatives).
Etiologic pre­ The proportion of animals with positive test
dictive value* results that truly have the disease of interest,
as opposed to carriage of an organism in
the absence of disease.
*Gunnarson, RK, Lanke J. The predictive value of microbiologic
diagnostic tests if asymptomatic carriers are present. Statist Med 2002;
21:1773–1785.
results due to low analytical specificity are more likely when
testing animals from populations where the prevalence of true
positive test results is low. In this situation, a test has a low posi-
tive predictive value, and confirmatory testing may be required.
A quality assurance program should be in effect within the labo-
ratory to ensure that specimens submitted are of adequate qual-
ity, that sufficient technical expertise is present to perform the
tests, and that appropriate control assays are included with each
run to ensure proper assay performance.
Negative Results for Antigen Detection
Negative results for antigen will occur if the antigen is not present
in the specimen being tested. They may also occur if the amount
of antigen is below the limit of detection of the assay (e.g.,
low worm burden in heartworm disease). For some infectious
agents, such as FIV, antigen testing is not performed because
antigen levels during infection are typically extremely low. The
amount of antigen present can also vary over the course of a
particular infection. Finally, false-negative results may occur
when antibody present in the specimen complexes antigen and
makes it unavailable for detection by the immunoassay.
Positive Results for Antigen Detection
Positive results for antigen can indicate the presence of the sus-
pected pathogen in the specimen. They do not necessarily imply
that the pathogen is the cause of an animal’s clinical signs. Anti-
gen can also be present in the absence of intact, viable organisms.
Positive results can follow recent vaccination with the pathogen
of interest. For example, positive fecal parvovirus antigen tests
can occasionally occur in fecal specimens obtained several days
after attenuated live virus vaccination of kittens for feline panleu-
kopenia virus.6 False positives can occur as a result of antigenic
cross-reactivity with some other substance or organism in the
specimen. An example is positive serum and urine antigen assays
for Aspergillus spp. in dogs treated with Plasmalyte infusions or
those infected with other molds such as Paecilomyces spp.7
Negative Results for Antibody Detection
Negative results for antibody will occur if the patient has not
been exposed to the infectious agent of interest. They may also
occur if the patient has not yet had time to develop antibod-
ies. For example, negative antibody tests early in the course of
illness are common with leptospirosis and canine granulocytic
anaplasmosis. In this situation, a convalescent titer is required
for diagnosis using serology. Negative antibody test results
can occur in severely immunosuppressed animals, such as in
cats with advanced FIV infection that no longer can mount an
immune response, or cats with advanced feline infectious peri-
tonitis. Negative results may also occur because the test lacks
sensitivity (i.e., low analytical sensitivity).
Positive Results for Antibody Detection
Positive results for IgG antibody suggest previous exposure
to a pathogen and do not necessarily indicate active infection.
Paired IgG titers are required to document recent infection.
Single positive results for IgM-specific assays may indicate a
recent infection or a chronic infection that continues to stimu-
late the immune system. Previous immunization may also result
in positive results. If the infection is chronic and persistent, such
as with FIV infection, positive results indicate active infection,
provided there has been no history of previous FIV vaccina-
tion. False-positive results for antibody may also occur when
antigenic cross-reactivity occurs. For example, dogs recovering
from Leptospira infection have been shown to test positive on
IFA for the related spirochete B. burgdorferi.
REFERENCES
1. Levy J, Crawford C, Hartmann K, et al. 2008 American Association
of Feline Practitioners’ feline retrovirus management guidelines.
J Feline Med Surg. 2008;10(3):300-316.
2. Leschnik MW, Kirtz G, Khanakah G, et  al. Humoral immune
response in dogs naturally infected with Borrelia burgdorferi sensu
lato and in dogs after immunization with a Borrelia vaccine. Clin
Vaccine Immunol. 2010;17(5):828-835.
3. Pomrantz JS, Johnson LR, Nelson RW, et al. Comparison of sero-
logic evaluation via agar gel immunodiffusion and fungal culture
of tissue for diagnosis of nasal aspergillosis in dogs. J Am Vet Med
Assoc. 2007;230(9):1319-1323.
4. Pappagianis D, Zimmer BL. Serology of coccidioidomycosis. Clin
Microbiol Rev. 1990;3(3):247-268.
5. Spector D, Legendre AM, Wheat J, et  al. Antigen and antibody
testing for the diagnosis of blastomycosis in dogs. J Vet Intern
Med. 2008;22(4):839-843.
6. Patterson EV, Reese MJ, Tucker SJ, et  al. Effect of vaccina-
tion on parvovirus antigen testing in kittens. J Am Vet Med Assoc.
2007;230(3):359-363.
7. Garcia RS, Wheat LJ, Cook AK, et al. Sensitivity and specificity of
a blood and urine galactomannan antigen assay for diagnosis of
systemic aspergillosis in dogs. J Vet Intern Med. 2012;26:911-919.
CHAPTER 3
Isolation and Identification of Aerobic
and Anaerobic Bacteria
Jane E. Sykes and Shelley C. Rankin
w KEY POINTS
• B  ecause of the increasing prevalence of antimicrobial drug
resistance among bacteria isolated from dogs and cats, culture
and susceptibility is always recommended when a bacterial
infection is suspected at a normally sterile anatomic site.
• Successful isolation of aerobic and anaerobic bacteria from a
clinical specimen relies on collection of a sufficient quantity
of material, from the correct location, and appropriate storage
and transport of the specimen to the laboratory. Care should be
taken to prevent specimen contamination during collection.
• The laboratory should be provided with an appropriate history
if fastidious, anaerobic, or slow-growing bacterial organisms
are suspected.
INTRODUCTION
Factors that influence the detection of clinically relevant organ-
isms in specimens collected from dogs and cats are:
1. An appropriate level of suspicion for the presence of a bacterial
infection;
2. Development of a list of differential diagnoses that reflects
the types of bacterial pathogens that might be the cause of
clinical signs, because of the requirement of some bacteria for
special transport conditions, culture conditions, or prolonged
incubation;
3. Collection of a sufficient amount of specimen;
4. Collection of the correct type of specimen;
5. Specimen collection from the correct anatomic location; and
6. Proper handling, storage and transport of the specimen, which
should be sent with a request for culture to the laboratory
within acceptable time limits.
These factors cannot be overemphasized. Without them, time and
money invested in collection of specimens and culture is wasted.
Given the increasing prevalence of multidrug resistance among
bacteria that infect dogs and cats, veterinarians should make
attempts to confirm a bacterial infection at a normally sterile site
by requesting microscopic evaluation of direct smears and culture
by a laboratory whenever possible before the choice is made to
administer an antimicrobial drug.
The purpose of this chapter is to provide information in regard
to the correct way to collect and handle specimens for culture
before they are sent to the laboratory, and to outline bacteriologic
procedures performed in the microbiology laboratory that are of
relevance to the small animal clinician.
Specimen Collection and Transport
Veterinary clinicians should collect specimens for culture
using an appropriate method from the correct anatomic site.
• C  oncurrent examination of a Gram-stained smear by the labo-
ratory assists in assessment of specimen quality and interpreta-
tion of culture results.
• Antimicrobial susceptibility testing is most commonly per-
formed using broth microdilution or agar diffusion testing and
aims to determine the minimum concentration of an antimi-
crobial drug that inhibits growth of the organism in vitro (mini-
mum inhibitory concentration, or MIC). The laboratory uses
MIC breakpoints, which are established and revised regularly
by national standards agencies, to assess the susceptibility of
an organism to a particular antimicrobial drug.
Collection of a sufficient amount of specimen is very important.
Table 3-1 suggests optimal specimen types for detection of bac-
teria from specific anatomic sites. Attempts should always be
made to prevent contamination with commensal bacteria during
specimen collection. When anaerobes are suspected, aspirates or
biopsies should be obtained rather than swabs, and the speci-
men should be stored at room temperature and not refrigerated.
This is because oxygen diffuses into cold specimens more rap-
idly than specimens held at room temperature.
Specimens should be transported to the laboratory imme-
diately. As a general rule for aerobic culture, if transport
requires longer than 2 hours, appropriate transport medium
should be used or specimens should be refrigerated without
transport medium. Although specimens can be held for up to
72 hours, culture of specimens older than 24 hours should
be avoided, even if they have been refrigerated or placed
in transport media, because of increased likelihood of false
positive and false negative results with longer storage times.
Transport medium for bacterial culture is usually supplied in
a plastic tube with one or two swabs attached to the tube’s
cap (Figure 3-1). The medium is designed to maintain bac-
terial viability without causing significant growth. It usually
consists of a small amount of agar, reagents that maintain pH,
a colorimetric pH indicator that indicates whether oxidation
has occurred, and specific factors that maintain the viability
of certain pathogens. Some organisms, such as chlamydiae
and leptospires, have specific transport medium requirements.
Specimens that are placed in transport media should not be
refrigerated.
Specimens should be labeled with the patient’s name, patient
number, source of specimen, and the date and time of collection,
and should be labeled, packaged, and transported according to
regional and national regulations (see Chapter 1). Specimens
for anaerobic culture should preferably be transported in an
oxygen-free container.
17
18 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

TABLE 3-1
Suggested Specimen Types for Bacterial Culture from Selected Anatomic Sites and Likely Pathogens Present
Specimen Type Suggested Volume for Culture Likely Pathogens Present
Blood ≥10 mL for patients weighing >10 kg, or 1% Gram-positive and gram-negative aerobes. Common contam-
of patient blood volume inants are coagulase-negative staphylococci, Corynebacte­
rium, Bacillus spp., and propionibacteria (see Chapter 86)
CSF ≥0.5 mL Gram-positive and gram-negative aerobes, anaerobes,
­Brucella spp. (dogs), Nocardia, Actinomyces,1 Myco­
plasma spp. (cats)2 (see Chapter 90)
Middle ear Aspirate of fluid or pus through tympanic Gram-positive and gram-negative aerobes, anaerobes,
membrane ­Mycoplasma spp., Actinomyces spp.2,3 (see Chapter 84)
Skin Swab from epidermal collarette or ruptured pustule Gram-positive aerobes (especially staphylococci), rarely
(superficial pyoderma); biopsy from which dermis Pseudomonas aeruginosa (see Chapter 84)
has been removed (deep pyoderma)
External ear Swab ear canal after removing debris and crusts Gram-positive aerobes, Pseudomonas aeruginosa (see
from the canal Chapter 84)
Cornea Scrape or swab exposed corneal stroma and inocu- Gram-positive aerobes, Mycoplasma spp., Pseudomonas
late directly onto media. Referral to veterinary aeruginosa4
ophthalmologist recommended
Abdominal fluid 1-5 mL Gram-negative and gram-positive aerobes, anaerobes,
Actinomyces spp. (see Chapter 88)
Pleural fluid 1-5 mL Gram-negative and gram-positive aerobes, anaerobes,
Actinomyces spp., Nocardia spp., Mycoplasma spp.
(see Chapter 87)
Feces Fresh whole stools preferred. Avoid swabs or fecal Escherichia coli, Salmonella, Clostridium perfringens,
loops containing fecal matter due to low specimen Clostridium difficile, Campylobacter spp., Anaerobio­
size. Submit in clean, dry leak-proof container. spirillum (Chapters 45-49 and 88)
Urine 1-5 mL urine collected using cystocentesis Gram-negative and gram-positive aerobes, Coryne­bacterium
urealyticum (see Chapter 89)
Bronchoalveolar 1-5 mL Gram-positive and gram-negative aerobes, anaerobes
lavage (aspiration pneumonia), Mycoplasma spp., rarely Myco­
specimens bacterium spp. (see Chapter 87)

results of culture and susceptibility testing. If sufficient material

FIGURE 3-1  Swab system containing transport media for aerobic bacterial culture.
Diagnostic Methods
Microscopic Examination of Direct Smears
A Gram stain prepared from the specimen can permit the rapid
preliminary diagnosis of infection and determine whether the
organism(s) present are gram-positive or gram-negative (Box
3-1; Figure 3-2). This helps guide the clinician to select an
appropriate empiric therapy, if necessary, while awaiting the
is available, examination of a direct smear also helps to deter-
mine whether a specimen is adequate for culture and aids inter-
pretation of culture results. For swab or aspirate specimens,
clinicians should consider providing a separate specimen for a
direct smear in addition to a specimen for culture.
Acid-fast stains can be used on smears to assist in the identifi-
cation of Nocardia and Mycobacterium spp., as well as on fecal
smears to detect some parasitic oocysts, such as those of Cryp­
tosporidium. These organisms stain magenta as a result of their
high cell wall mycolic acid content, which in turn results in their
resistance to decolorization after staining (which is often per-
formed using an acid-alcohol solvent, hence the term acid-fast).
Examples of acid-fast staining methods are the Ziehl-Neelsen,
Fite’s, and Kinyoun stains.
Other microscopic techniques used for bacterial identifica-
tion in specialized laboratories include darkfield microscopy,
phase-contrast microscopy, and fluorescence microscopy (see
Chapter 2). Darkfield and phase contrast microscopy allow visu-
alization of organisms without fixation and staining, so motility
as well as structure can be assessed. In darkfield microscopy, the
 CHAPTER 3  Isolation and Identification of Aerobic and Anaerobic Bacteria 19

condenser only permits light that bounces off an object to pass

BOX 3-1
Procedure for Gram Staining
Heat-fix specimen on the slide over a gentle flame or fix in
95% methanol for 2 min, and then air dry
Flood with crystal violet solution for 15 to 60 s (10 g of
90% dye in 500 mL absolute methanol)
Wash with water
Flood with iodine for 15 to 60 s (6 g of I2 and 12 g of KI
in 1800 mL of water)
Decolorize with acetone-alcohol (400 mL of acetone in
1200 mL of 95% ethanol)
Wash immediately
Counterstain with safranin for 15 to 60 s (10 g of dye in
1L of water)
Wash, blot dry, and examine using light microscopy
FIGURE 3-2  Gram-stained smear of gram-positive cocci (dark blue) and gram-­
negative rods (red) (1600× oil magnification).
from below up into the objective, so the object appears bright
against a dark background. Darkfield microscopy is used pri-
marily for identification of fine spirochetes such as leptospires,
which cannot be visualized using conventional microscopy (see
Chapter 50). In phase-contrast microscopy, slowing and deflec-
tion of beams of light occur as they pass through an object.
Bacterial Culture
The performance of bacterial culture for diagnostic purposes
requires appropriate laboratory facilities, proper biosafety
level-2 containment, properly trained individuals, reference
strains for quality control testing, and the use of standard pro-
tocols.5 Media used for bacterial culture may be categorized as
general-purpose, enriched, selective, differential, and specialized
media (Table 3-2). Some media belong to more than one of these
categories. For example, MacConkey agar is both a selective and
a differential medium (Figure 3-3). Media for culture contains a
nutrient source, most commonly peptones (hydrolyzed animal
or vegetable substances), and is adjusted to a specific pH. Solid
medium contains agar, which is derived from seaweed. Other
nutrients may be added based on specific organism requirements.
More than 100 different types of bacteriology media exist, most
of which can be purchased as preprepared plates or broth.
Blood culture media contain the anticoagulant sodium poly-
anethol sulfonate (SPS), which is less inhibitory to bacterial
growth than other anticoagulants. Some blood culture media
(e.g., BD BACTEC Plus) contain resin beads that bind antimicro-
bial drugs in the patient’s blood specimen, so they do not inhibit
bacterial growth (Figure 3-4). The detection of bacterial growth
in blood culture systems can be automated using instrumentation
that measures emission of fluorescence or a colorimetric change
in a sensor. The change occurs as CO2 concentrations within
the bottle increase, which indicates the presence of bacterial
growth. Some instruments detect pressure changes in the head
of the bottle. The culture is monitored at regular intervals by
the instrument, to optimize early detection of bacterial growth.
Positive blood culture bottles are then evaluated by performing
a Gram stain of the broth, and subculture to appropriate media.

TABLE 3-2
Types of Media Used in Bacteriology
Medium Type Attributes Example
Transport Nonnutritive liquid or semisolid medium. Promotes Amies transport medium
­organism viability without allowing significant
growth.
General-purpose Allows growth of most aerobic and facultatively Sheep blood agar
­anaerobic organisms
Enriched Promotes growth of fastidious organisms that require Chocolate agar, which is enriched with heat-
specific nutrients, for example Francisella treated blood, which gives it a brown color
Selective Selects for growth of certain organisms while inhibiting MacConkey agar, which selects for gram-­
others negative bacteria
Differential Aids in preliminary identification of organisms based MacConkey agar, which promotes pink colony
on colony appearance formation by organisms that ferment lactose
Specialized Selects for a specific pathogen through the addition of Anaerobic medium, which contains hemin,
specific nutrients vitamin K, and reducing agents
20 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
Another blood culture system is the Isolator lysis centrifuga-
tion system (DuPont, Wilmington, DE) (See Figure 3-4, B). In
this system, blood is added to a tube that contains anticoagulant
and a reagent that induces RBC lysis. The tube is centrifuged,
and the pellet is plated onto agar media and incubated. This
method allows colony counts to be obtained. The tubes allow
isolation not only of aerobes, but also some anaerobic bacteria.
In the laboratory, media for bacterial culture should be
warmed to room temperature before inoculation, and dam-
aged media, such as dehydrated medium or medium that has
changed color, should not be used. Medium is stored in the dark
at 2°C to 8°C and discarded when it reaches its expiration date.
After plating, incubation is most commonly performed at 35°C
to 37°C in air for a minimum of 2 days. Conditions vary for
specific bacteria, and growth in 3% to 5% CO2 is preferred by
some fastidious organisms. The laboratory should be informed,
FIGURE 3-3  Growth of bacteria on MacConkey medium that shows the selective and
differential qualities of the medium. The medium contains substances that inhibit the
growth of gram-positive bacteria. By using the lactose available in the medium, bacteria
such as Escherichia coli, Enterobacter spp., and Klebsiella spp. produce acid when they grow
on the medium, which lowers the pH of the agar and results in the formation of red to
pink colonies.
A B
FIGURE 3-4  Blood culture systems. A, BACTEC system showing resin beads that bind
antimicrobial drugs. B, Pediatric, aerobic, and anaerobic BACTEC systems for blood culture
together with adult and pediatric WAMPOLE Isolator tubes (front) for lysis-centrifugation
culture of blood.
by providing an adequate clinical history, when pathogens with
special culture requirements are suspected, such as Campylo­
bacter, Mycoplasma, Actinomyces, Nocardia, and Mycobacte­
rium spp. These organisms require special media or prolonged
incubation times (e.g., weeks). Specimens for anaerobic culture
should be plated onto appropriate media immediately and
incubated in an anaerobic environment. Anaerobic containers,
which create an anaerobic environment through the use of gas-
generating reagents, can be used for incubation (Figure 3-5).
Because anaerobes grow more slowly than aerobes, anaerobic
cultures should be held for 7 days before being reported as
negative.
Blood and Intravascular Catheters
Bacteremic animals may be continuously or intermittently bac-
teremic. Because of intermittent bacteremia, multiple, separate
specimens, each containing greater than or equal to 10 mL of
blood, are recommended. Pediatric bottles are available that
can be inoculated with as little as 1.5 mL of blood, although
the chance of a positive isolation increases as the blood volume
drawn increases. Therefore, as much blood should be drawn as
possible, provided that collection of a large volume of blood
does not jeopardize the patient. Bottles should be held at room
temperature and submitted immediately to the laboratory.
Two, and ideally three, blood samples should be collected using
meticulous sterile technique, and the timing of blood draws
should depend on the severity of the animal’s illness (Box 3-2).
Inoculated bottles are incubated in aerobic conditions for 7 days
before being reported as negative. Additional blood samples are
not recommended, because they add cost without a significant
increase in sensitivity.6 The choice to inoculate anaerobic blood
culture bottles is controversial. Studies in humans have shown
that the prevalence of anaerobic bloodstream infections has
decreased, and routine inoculation of anaerobic blood culture
bottles may not be necessary. In one veterinary study, anaerobes
were not identified in any of 33 dogs with endocarditis using
FIGURE 3-5  System for isolation of anaerobic and microaerophilic bacteria. The
packets are opened, and the sachets inside are placed into the container with the inocu-
lated plates. The container is sealed immediately. The sachets contain ascorbic acid.
­Atmospheric oxygen in the container is rapidly absorbed and replaced with carbon dioxide
in an ­exothermic reaction process. After an appropriate incubation period, the container is
opened and the plates inspected for growth. An anaerobic indicator can be placed within
the box to ensure that the proper conditions are maintained.
 CHAPTER 3  Isolation and Identification of Aerobic and Anaerobic Bacteria
anaerobic culture of blood, but anaerobic bacteremia may be
occur with other underlying causes of bloodstream infections.6
Common blood culture contaminants include coagulase-negative
staphylococci, Bacillus spp., corynebacteria, and propionibac-
teria. When only one blood culture is positive for one of these
organisms, contamination is likely, but the presence of mul-
tiple positive blood cultures from separate sites suggests true
bacteremia.
Culture of intravascular catheter tips is performed to deter-
mine whether a catheter is a source of bacteremia and should
only be performed simultaneously with blood culture. The cath-
eter is removed after cleaning the skin around the catheter site
with alcohol, and the distal 5 cm is aseptically removed into a
sterile tube and submitted immediately.7 In the laboratory, the
catheter tip is rolled onto an agar plate. The result of catheter
culture alone should not change treatment. Therefore, in most
situations, blood culture may be all that is required.
Skin
For dogs with superficial pyoderma, specimens for culture
should ideally be collected from a pustule. No surface disinfec-
tion should be performed. The hair should be clipped using ster-
ile scissors; clippers should be avoided. A pustule is lanced with
a sterile narrow-gauge needle, and pus on the needle or exuding
from the pustule is applied to a sterile swab, which can then be
placed in transport medium for transport to the laboratory. If a
pustule cannot be found, cultures can also be obtained by lift-
ing crusts and swabbing beneath the crust, or rolling the swab
across an epidermal collarette two to three times. Papules can be
cultured by biopsy after surface disinfection with 70% alcohol,
BOX 3-2
Suggested Procedure for Obtaining Blood Cultures
Shave site.
Clean skin with 10% povidone iodine or 1-2% tincture of
iodine, swabbing concentrically from the center; allow
to dry (wait 1 min).
If a catheter must be used for specimen collection, clean
access port with 70% alcohol and allow to dry. Avoid
collection from a catheter if possible.
Clean stopper on top of the culture tube or bottle with
70% alcohol; allow to dry (wait 1 min).
Perform venipuncture using sterile gloves to palpate the vein.
Inoculate blood culture bottle without changing the needle.
Space cultures based on severity of illness, before starting
antimicrobial therapy.
• Acute febrile illness: antimicrobials need to be
changed or started immediately: two sets from
separate sites, all within 10 minutes. Consideration
could be given to drawing a third set using a resin
bottle just before administering the second dose of
antimicrobials (i.e., when trough drug concentra-
tions are present)
• Acute endocarditis: 3 sets from 3 separate sites,
within 1-2 hours
• Nonacute disease: 3 sets from separate sites within
24 hours, spaced at intervals of at least 3 hours
21
allowing the surface to dry, and use of a 3- to 4-mm punch
and sterile surgical instruments to collect the biopsy, which is
then submitted in transport medium or a small amount of sterile
saline. The biopsy site can then be sutured closed.
For deep pyoderma, a biopsy of the lesion should also be
obtained after surface disinfection with 70% alcohol. The der-
mis should be excised with a sterile blade and the biopsy sub-
mitted as just described.
For dogs and cats with wounds, aspirates and deep tissue
biopsies after surgical dissection are always preferable to swab
specimens. Swab specimens are often contaminated by surface
microorganisms, contain only a small amount of material, and
are not amenable to optimal anaerobic transport. For this rea-
son, collection of pus from fistulous tracts using a swab should
also be avoided. If an abscess is present, an aspirate of the
abscess fluid should be collected and submitted for aerobic and
anaerobic culture. For dogs and cats with cellulitis, an aspirate
can be attempted by injecting a small volume of sterile saline
into the lesion and aspirating back into a syringe, although
bacterial yields may be low and biopsy may be required in
some cases.
Body Fluids
Usually, 1 to 5 mL of body fluid is required for culture. Fluid
can be inoculated into blood culture bottles in the laboratory or
alongside the patient. Body fluids that have the potential to clot
should be submitted to the laboratory in a tube that contains
an appropriate ratio of anticoagulant. In the laboratory, body
fluids with volumes that exceed 1 mL are generally concentrated
using centrifugation and resuspension of the pellet in a smaller
volume (e.g., 0.5 mL) of supernatant before they are inoculated
onto agar.
For cerebrospinal fluid, the chance of organism isolation also
increases when the volume submitted is maximized. If possible,
at least 0.5 mL should be submitted for culture. Cerebrospinal
fluid specimens should be immediately transported to the labo-
ratory, preferably at room temperature.
Tissue Specimens
Attempts should be made to obtain as large a tissue specimen as
possible for culture. The use of swabs to collect specimens for cul-
ture during surgical procedures is discouraged because of insuf-
ficient specimen size. Small pieces of tissue can be submitted in
a small amount of sterile, nonbacteriostatic saline. In the labora-
tory, the tissue is minced using sterile procedures to release organ-
isms before smears are prepared and media inoculated. Because
an anaerobic environment can be maintained within pieces of tis-
sue greater than or equal to 1 cm3 in volume, these are suitable
for anaerobic culture without oxygen-free transport. Such tissue
specimens may be stored at room temperature for up to 24 hours.
Airway Specimens
Specimens from the upper respiratory tract are generally con-
taminated with commensal bacteria. Aerobic bacterial culture
of a nasal wash specimen may be indicated in cats with chronic
rhinosinusitis in which an antimicrobial resistant organism is
suspected, in particular Pseudomonas aeruginosa. Isolation of
Bordetella bronchiseptica may also have clinical significance
in dogs and cats with nasal discharge. Growth of Mycoplasma
spp. from the nasal cavity has uncertain clinical significance,
because Mycoplasma spp. can be isolated from the nasal cavities
of both healthy cats and cats with rhinosinusitis.8-11
22 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
Specimens from the lower respiratory tract may be collected
using transtracheal, endotracheal, or bronchoalveolar lavage.
Bronchial brush specimens may also be submitted for culture,
after placing them in 1 mL of sterile nonbacteriostatic saline.
The presence of intracellular bacteria on cytologic examination
is consistent with a bacterial infection, rather than contamina-
tion. Anaerobic culture could be considered for animals with
aspiration pneumonia or migrating plant awn foreign body
pneumonia.
Feces
Whole stools or swabs that contain fecal matter should be pro-
cessed within 1 to 2 hours or refrigerated as soon as possible
after specimen collection and submitted within 24 hours. The
specimens should not be contaminated with barium or urine.
Rectal swabs that contain fecal material can be immersed in
transport medium until processing. Testing of more than one
sequential specimen may improve detection of fecal pathogens
due to the low sensitivity of fecal culture methods. Selective
media are required for isolation of Salmonella spp. Because
Clostridium perfringens and C. difficile are commonly detected
in normal stool specimens, toxin antigen testing may be more
appropriate than culture to determine the role these organ-
isms may be playing in diarrhea (see Chapter 48). Special
medium is also required for isolation of Campylobacter (see
Chapter 47).
Urine
Urine should be collected using cystocentesis whenever possi-
ble. Collection of urine via sterile catheterization in male dogs
is acceptable when cystocentesis is not possible. Quantitative
cultures should always be performed. For specimens obtained
by cystocentesis, any level of bacterial growth is significant,
although most urinary tract infections contain greater than
or equal to 103 colony-forming units (CFU)/mL of bacteria.
For urine specimens obtained via catheter from male dogs,
bacterial growth of greater than or equal to 105 CFU/mL has
been considered significant.12 Only aerobic bacterial culture is
routinely indicated. Anaerobic urine culture may be indicated
when there is radiologic evidence of emphysematous cystitis.
Urine specimens should be refrigerated and submitted to the
laboratory as soon as possible. Special urine transportation
tubes can be used to preserve bacteria within the specimen
provided they are appropriate for the volume of urine col-
lected (e.g., BD Vacutainer urine culture and sensitivity tube).
Urine “paddles” are also available for in-house veterinary use.
These allow inexpensive screening for the presence of absence
of infection (­Figure 3-6). The paddles have media impregnated
on them, are designed to be incubated in the clinic, and can
provide some information on the infecting bacterial species
present and the number of CFU/mL. Positive paddles can then
be submitted to the laboratory for culture and susceptibility
testing, although this does not always permit accurate iden-
tification of all bacteria present in mixed infections (see also
Chapter 89).
For animals with a urinary catheter in place and a suspected
urinary tract infection, culture of the catheter tip is not recom-
mended, because it is generally contaminated with commensal
organisms from the urethra. Instead, a urine specimen should
be obtained by aspiration from a new urinary catheter, or, if
feasible, using cystocentesis. If a catheter is used to collect the
specimen, several milliliters of urine should be removed and
FIGURE 3-6  Media paddle available for screening urine for the presence of an aerobic
bacterial infection. The paddle is impregnated on each side with selective and nonselec-
tive media (in this case eosin methylene blue medium, shown, and cysteine lactulose
electrolyte-deficient media, on underside, respectively). The paddle is flooded with urine,
incubated, and inspected for growth. Eosin methylene blue medium is red and selects for
growth of gram-negative enteric organisms. Cysteine lactose electrolyte-deficient medium
is initially green but turns yellow in the presence of lactose-fermenting organisms.
discarded first to clear the catheter. Urine culture should never
be performed on collection bag specimens.
Fastidious and Unculturable Organisms
Some bacteria, such as Bartonella spp., Borrelia burgdorferi,
and Leptospira spp., require special culture conditions (see
Chapters 50 to 52). Others, such as hemotropic Mycoplasma
species, are unculturable, and DNA amplification methods such
as PCR are required for detection of these organisms. Cell cul-
ture is required to propagate certain bacteria. Examples include
Chlamydia felis and tick-borne pathogens such as Anaplasma
phagocytophilum.
Bacterial Identification
Following aerobic or anaerobic culture, presumptive bacterial
identification can be made on the basis of colony morphol-
ogy and microscopic morphology (e.g., Gram stain character-
istics, rods or cocci). Further differentiation is generally based
on a series of additional phenotypic tests, which are selected
based on microscopic morphology and whether the organism
is gram-negative, gram-positive, aerobic, or anaerobic. Micro-
biology laboratories use published methods to identify bacte-
ria.13 Preliminary tests used to identify gram-positive aerobes
include the presence or absence of hemolysis on a blood agar
plate (Figure 3-7), a positive or negative catalase test, and the
production of coagulase. Identification of gram-negative aer-
obes relies on whether or not the organism ferments lactose,
indole production, oxidase production, and the production of a
variety of other enzymes. Assessment of motility and the ability
to ferment carbohydrates are also used to differentiate bacteria.
Biochemical test strips are available that allow multiple tests
to be performed simultaneously (e.g., API Strips, bioMérieux)
(Figure 3-8), so that identification can be made within 24 to
48 hours. Automated identification systems that consist of a
96-well microtiter tray that contains a variety of biochemical
reagents (e.g., Sensititre, TREK Diagnostics, Cleveland) are also
in use in some veterinary diagnostic laboratories.
Molecular methods such as 16S rRNA gene PCR and sequenc-
ing are now being used to identify organisms to the species level
(see Chapter 5). Speciation of some mycobacteria requires analysis
 CHAPTER 3  Isolation and Identification of Aerobic and Anaerobic Bacteria
FIGURE 3-7  Mixture of beta-hemolytic and nonhemolytic enteric bacteria growing
on blood agar. Beta-hemolysis is characterized by complete clearing of the agar around
each colony.
of mycolic acid content using high-performance liquid chromatog-
raphy. Recently, the availability of matrix-assisted laser desorp-
tion ionization time-of-flight mass spectrometry instrumentation
(i.e. the Bruker Biotyper and VITEK MS systems) has revolution-
ized bacterial identification in the clinical microbiology labora-
tory. These systems use mass spectrometry to rapidly (on average
in 6 minutes) and accurately (often >95%) identify organisms to
the species level based on their chemical composition, provided a
reference spectrum for the organism is available in the database
used for comparison. Although the instrumentation is expensive,
the ultimate cost is lower than with conventional biochemical
methods, because of reduction in labor and reagent costs.14
Interpretation of Bacterial Culture Results
Specimens That Test Negative
Negative results (or lack of growth) using bacterial culture do
not imply the absence of bacteria. Negative test results can occur
as a result of inadequate specimen size (such as when swabs or
aspirates are submitted), when there are inadequate organism
numbers at the site of specimen collection, when overgrowth with
commensal organisms occurs, or when there is loss of organism
viability during transportation to the laboratory. The last is par-
ticularly problematic for anaerobic bacteria. In some situations,
multiple bacterial species are present within a specimen, and one
species (such as Proteus spp.) swarms the plate and obscures the
presence of another. The identification of multiple bacterial types
on a Gram-stained smear allows the laboratory to plan culture
methods that maximize detection of all species present.
Negative test results can also occur when the organism is
not inoculated onto the correct media or sufficient time is not
allowed for growth. Clinicians must consider whether fastidi-
ous or slow-growing organisms might be present and inform
the laboratory if they are suspected. Poor laboratory qual-
ity control procedures may also lead to false-negative test
results. Use of an accredited laboratory can help minimize such
problems.
Specimens That Test Positive
The detection of bacterial organisms within a specimen using
culture does not imply that the organism is the cause of an
23
animal’s clinical signs. Contamination is the most common
cause of false positive cultures. Isolation of only one or two
colonies of coagulase-negative staphylococci, Bacillus spp.,
Corynebacterium spp., or propionibacteria suggests contami-
nation. Isolation of large numbers of a single type of bacteria
from a normally sterile site is usually clinically significant, espe-
cially when supported by cytologic evidence of bacteria within
phagocytes.
Antimicrobial Susceptibility Testing
Importance of Antimicrobial Susceptibility Testing
Given the increasing recognition of antimicrobial resistance
among bacteria that are isolated from small animal patients,
determination of antimicrobial susceptibility can be as or more
important to the clinician than the identity of the infecting
agent. The result of antimicrobial susceptibility testing is gen-
erally available 2 to 3 days after submission of a specimen for
culture. In animals that are critically ill, empiric antimicrobial
therapy may already have been initiated by the time this result
is available. The susceptibility results may show that the organ-
ism is resistant to a drug being used, in which case the drug
should be changed to one to which the organism is susceptible.
The susceptibility pattern can help to identify an alternate drug
when the patient does not tolerate the initial drug prescribed.
Susceptibility testing may indicate that the organism is suscep-
tible to a more narrow-spectrum (and generally less expensive)
antimicrobial drug than the drug initially prescribed, in which
case the spectrum should be narrowed to minimize the develop-
ment of antimicrobial resistance.
Methods of Susceptibility Testing
Susceptibility testing can be performed using dilution methods
or diffusion methods. The minimum inhibitory concentration
(MIC) is the lowest concentration of antimicrobial drug that
inhibits visible growth of an organism over a defined incubation
period, most commonly 18 to 24 hours. The MIC is determined
using dilution methods, which involve exposing the organism to
twofold dilutions of an antimicrobial drug. The concentration
range used varies with the drug and the organism being tested.
In vitro factors that influence MIC include the composition of
the medium, the size of the inoculum, incubation conditions,
and the presence of resistant bacterial subpopulations within
the inoculum. Standard protocols are published by the Clinical
and Laboratory Standards Institute (CLSI) that specify medium
composition and pH, inoculum size, inoculation procedures,
agar depth, and incubation conditions, as well as quality control
requirements.13 Failure to comply with these protocols can lead
to erroneous results, so laboratories that adhere to CLSI stan-
dards should be used whenever possible. MIC testing does not
take into account in  vivo factors such as postantibiotic effect
and the effects of protein binding on antimicrobial drug activity
(see Chapter 6).
Dilution Methods
Dilution methods include broth macrodilution, broth micro­
dilution, and agar dilution. The most widely used method
in North America is broth microdilution, whereby twofold
dilutions of antimicrobials are made in a broth medium
in a microtiter plate (Figure 3-9). Pre-prepared frozen or
freeze-dried microtiter plates for inoculation are available
24 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

FIGURE 3-8  API bacterial identification strips. Only the left half of the reaction strip is shown. Top (api20STREP): Enterococcus faecium. Bottom (api20E): Escherichia coli. Positive reactions
are assigned a numerical code on the handwritten strip. The codes in each three-well group are added to generate a new (seven-number) code that can be used to identify the organism.

commercially (e.g., Sensititre plates, TREK Diagnostic Sys-
tems, Cleveland). The results can be determined using visual
examination of the plates for the inhibition of bacterial
growth, or by the use of semiautomated or automated instru-
mentation. Dilutions can also be performed in agar, each dilu-
tion being poured onto a plate in a standardized fashion and
allowed to set before inoculating it with the organism(s) of
interest. Agar dilution can be used to perform susceptibility
testing of fastidious bacteria with special medium require-
ments, such as Campylobacter spp.
The MIC for each antimicrobial drug tested against the
organism is reported to the clinician on the susceptibility panel.
It is the lowest concentration of antibiotic (usually in µg/mL)
that inhibits growth of the organism in  vitro. The lower the
 CHAPTER 3  Isolation and Identification of Aerobic and Anaerobic Bacteria 25

1 2 3 4 5 6 7 8 9 10 11 12
A AMP AMP AMP AMP AMP AMP AMP OXA+ OXA+ OXA+ OXA+ OXA+
0.25 0.5 1 2 4 8 16 0.25 0.5 1 2 4
B AUG2 AUG2 AUG2 AUG2 AMI AMI AMI AMI FOX FOX FOX FOX
4/2 8/4 16/8 32/16 4 8 16 32 2 4 8 16
C TIC TIC TIC TIC POD POD POD POD TIM2 TIM2 TIM2 TIM2
8 16 32 64 2 4 8 16 8/2 16/2 32/2 64/2
D SXT SXT SXT FOV FOV FOV FOV FOV FOV FAZ FAZ FAZ
0.5/9.5 1/19 2/38 0.25 0.5 1 2 4 8 4 8 16
E GEN GEN GEN GEN IMI IMI IMI IMI CLI CLI CLI CLI
1 2 4 8 1 2 4 8 0.5 1 2 4
F PEN PEN PEN PEN PEN PEN PEN PEN DOX DOX DOX POS
0.06 0.12 0.25 0.5 1 2 4 8 2 4 8
G XNL XNL XNL XNL XNL MAR MAR MAR MAR RIF RIF POS
0.25 0.5 1 2 4 0.25 0.5 1 2 1 2
H ENRO ENRO ENRO ENRO ERY ERY ERY ERY CHL CHL CHL POS
0.25 0.5 1 2 0.5 1 2 4 4 8 16

AMP, ampicillin; AUG2, amoxicillin/clavulanic acid (2:1 ratio); TIC, ticarcillin; SXT,
trimethoprim/sulfamethoxazole; GEN, gentamicin; PEN, penicillin; XNL, ceftiofur; ENRO, enrofloxacin;
FOV, cefovecin; AMI, amikacin; POD, cefpodoxime; IMI, imipenem; ERY, erythromycin; MAR,
marbofloxacin; OXA+, oxacillin + 2% NaCl; FOX, cefoxitin; TIM2, ticarcillin/clavulanic acid (constant 2);
CLI, clindamycin; DOX, doxycycline; CHL, chloramphenicol; FAZ, cefazolin; RIF, rifampin; POS, positive
control.
FIGURE 3-9  Broth microdilution for determination of MICs. A series of wells contain an antimicrobial drug at twofold dilutions in a standard format. One custom plate format is
shown (TREK Diagnostic Systems Sensititre Companion Animal MIC plate). A pellet of bacteria (in this case, Escherichia coli from an infected feline esophagostomy tube site) settles on the
bottom of each well when growth fails to be inhibited by the antimicrobial drug concentration tested in that well. The cutoff MICs that define susceptibility versus resistance are published
by the Clinical and Laboratory Standards Institute (CLSI) in the United States. This isolate was reported as resistant to ampicillin (>16 mg/mL), susceptible to amoxicillin-clavulanic acid
(8 mg/mL), susceptible to amikacin (8 mg/mL), susceptible to cefoxitin (8 mg/mL), resistant to ticarcillin (>64 mg/mL), susceptible to cefpodoxime (≤2 mg/mL), susceptible to ticarcillin-
clavulanic acid (16 mg/mL), resistant to trimethoprim-sulfamethoxazole (>2 mg/mL), susceptible to cefovecin (1 mg/mL), susceptible to cefazolin (≤4 mg/mL), susceptible to gentamicin
(2 mg/mL), susceptible to imipenem (≤1 mg/mL), resistant to doxycycline (>8 mg/mL), resistant to enrofloxacin and marbofloxacin (both >2 mg/mL), and resistant to chloramphenicol
(>16 mg/mL). The MIC for ceftiofur was ≤0.25 mg/mL, but because a breakpoint for this drug has not been published, only the MIC was reported, and not whether the isolate was sus-
ceptible or resistant. Results for some drugs tested, such as oxacillin, are not reported for gram-negative bacteria because these drugs are used to treat gram-positive bacterial infections.
26 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

MIC, the more potent is the antimicrobial at inhibiting bacterial combination, the organism is defined as susceptible. The break­
growth. point is not reported to the clinician. Breakpoints are established
on the basis of multiple factors (Box 3-3), which include (1) a
Diffusion Methods knowledge of MIC distributions and resistance mechanisms for
each organism-drug combination (Figure 3-11),16 (2) clinical
Diffusion methods include gradient diffusion (also known as response rates in humans and animal models, (3) how the drug
Etest) and disc diffusion. The Etest involves use of a plastic strip is distributed and metabolized in the body (pharmacokinetics),
coated with an antimicrobial gradient on one side and an MIC and (4) whether the drug is concentration dependent or time
interpretive scale on the other side (Figure 3-10, A). An agar dependent as it relates to antibacterial effect (pharmacodynam-
plate is inoculated with the organism of interest so that sub- ics) (see Box 3-3).15 Zone diameter breakpoints for disc ­diffusion
sequent growth of the organism forms a “lawn,” rather than testing are determined by correlation with MIC values. For
individual colonies (i.e., inoculation to confluence). At the time simplicity, breakpoints are established for antimicrobial drug
of inoculation, the strips are applied to the surface of the plate.
The antimicrobial drug diffuses into the medium, resulting in an
elliptical zone of growth inhibition around the strip. The MIC is
read at the point of intersection of the ellipse with the MIC scale
on the strip. Although the strips are expensive, Etests have the
advantage of being adaptable to use with fastidious organisms
and anaerobes.
Disk diffusion, also known as Kirby-Bauer antibiotic testing,
involves application of commercially available drug-impreg-
nated filter paper discs to the surface of an agar plate that has
been inoculated to confluence with the organism of interest.
Commercially available, mechanical disk-dispensing devices can
be used to apply several disks simultaneously to the surface of
the agar. The drug diffuses radially through the agar, the con-
centration of the drug decreasing logarithmically as the distance
from the disk increases. This results in a circular zone of growth
inhibition around the disk, the diameter of which is inversely
proportional to the MIC (see Figure 3-10, B). The zone diam-
eters are interpreted on the basis of guidelines ­published by A
CLSI (see Definition of Breakpoints, next), and the organisms
are reported as susceptible, intermediate, or resistant. Disk dif-
fusion can only be used to test rapidly growing organisms that
have consistent growth rates, for which criteria for interpreta-
tion of zone sizes are available.

Definition of Breakpoints
Once susceptibility testing has been performed, organisms are
classified on the susceptibility panel report as “susceptible” (S),
“resistant” (R), and, in some cases, of “intermediate” (I) sus-
ceptibility. This refers to a predicted in  vivo situation, rather
than in vitro susceptibility. The growth of “susceptible” isolates
is predicted to be inhibited by antimicrobial drug concentra-
tions that are usually achievable in blood and tissues using
normal dosage regimens. “Intermediate” isolates have MICs
that approach usually attainable blood and tissue levels and
for which response rates may be lower than those of suscep-
tible isolates. This category implies clinical efficacy in body sites
where the drugs are normally concentrated (e.g., enrofloxacin
and amoxicillin in urine) or when a higher-than-normal dose of
a drug can be used. “Resistant” isolates are predicted to grow
in the face of the usually achievable concentrations of the drug B
in blood and tissues.15
So why can the laboratory predict in vivo susceptibility on FIGURE 3-10  Alternatives to broth microdilution for antimicrobial suscepti­
the basis of MIC determination, which is an in  vitro test? In bility ­testing. A, Etest that shows susceptibility of a Bacteroides fragilis isolate to
­amoxicillin-clavulanic acid. The strip in the center is impregnated with a gradient of
order to do this, the laboratory refers to breakpoints, or clinical
­antimicrobial drug (in this case, amoxicill-clavulanic acid), with the highest concen-
­cutoff MICs (or, for disc diffusion testing, cutoff zone diam- tration at the top of the strip. The MIC is read at the point where bacterial growth is no
eters), which are established, published, and revised regularly longer inhibited by the strip (0.19 mg/mL in this case). B, Disc diffusion (also known as
by committees that are affiliated with standards agencies such as Kirby-Bauer testing) for Escherichia coli showing varying degrees of resistance to different
the CLSI. If the MIC determined in the microbiology laboratory antimicrobial drugs, reflected by zones of growth inhibition around discs containing each
is lower than a published breakpoint for that organism-drug antimicrobial drug.
 CHAPTER 3  Isolation and Identification of Aerobic and Anaerobic Bacteria
concentrations in the bloodstream, and are based on a ­specific
dosage regime for the antimicrobial drug tested. This dosage
regime is selected by the standards agency involved. Because
some antimicrobials are concentrated extensively in urine, some
veterinary laboratories may offer urine MIC ­panels, which
provide breakpoints for lower urinary tract infections. These
breakpoints are higher than corresponding serum MIC break-
points, so a greater proportion of organisms tested are classified
BOX 3-3
Factors Used by Standards Agencies to Establish
Breakpoints for Antimicrobial Susceptibility Testing
Bacterial Factors
• Evaluation of MIC distributions for each bacterial
species. These tend to be normally distributed (see
Figure 3-10; available for different antimicrobial
drugs at www.eucast.org).15
• Knowledge of bacterial resistance mechanisms, as
determined using phenotypic or genetic testing (e.g.,
testing for altered penicillin binding proteins in
staphylococci)
Pharmacokinetics of the antimicrobial drug when used at
a specific dosage regime in the animal species of interest,
including protein binding, taking into account variation
among individuals
Pharmacodynamic indices (e.g., whether killing is concen-
tration or time dependent) for the antimicrobial drug as
they relate to organism killing (see Chapter 6)
Clinical response rates in humans and animal models,
which are based on prospective, randomized controlled
clinical studies with well-defined dosage regimes
FIGURE 3-11  Histogram that shows the distribution of amikacin minimum inhibitory concentrations (MICs) for isolates of Escherichia coli. The MICs are normally distributed. (From
www.eucast.org. Accessed May 11, 2012.)
27
as “susceptible.” Urine MIC panels have been controversial
because the possibility of concurrent pyelonephritis (tissue drug
concentrations rather than urine concentrations) cannot always
be ruled out. Breakpoints may be reevaluated when new mecha-
nisms of resistance appear in bacteria or when new data are
generated that improve understanding of the pharmacokinetics
and pharmacodynamics of an antimicrobial drug.
For infections in sites such as the central nervous system, the
clinician needs to consider whether or not (1) an antimicrobial
will penetrate that site, (2) the drug is appropriate and safe, and
(3) a higher dose is necessary to achieve adequate concentra-
tions. The clinician must also consider factors such as immuno-
suppression and other concurrent illness or drug therapy when
treating infections on the basis of antimicrobial susceptibility
test results.
The bacterial species isolated determines which antimicro-
bials the laboratory selects for inclusion in susceptibility pan-
els. For example, susceptibility to cephalosporins may not be
reported for enterococci because of intrinsic resistance. Some
antimicrobial drugs (e.g., vancomycin, linezolid, imipenem)
should be reserved for treatment of multiple-drug resistant
organisms that cause life-threatening infections. Consultation
with a health professional with expertise in infectious diseases
or antimicrobial pharmacology is recommended before these
drugs are selected.
Minimum and Serum Bactericidal Concentration
The minimum bactericidal concentration (MBC) is the mini-
mum concentration of an antimicrobial drug that is bacteri-
cidal. It is determined by re-culturing (subculturing) broth
dilutions that inhibit growth of a bacterial organism (i.e., those
at or above the MIC). The broth dilutions are streaked onto
agar and incubated for 24 to 48 hours. The MBC is the low-
est broth dilution of antimicrobial that prevents growth of the
organism on the agar plate. Failure of the organism to grow
on the plate implies that only nonviable organisms are present.
The use of MBCs has been advocated by some for treatment
28 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
of serious infections (such as endocarditis) or for treatment of
immunosuppressed patients. Their value has been controver-
sial, and they are not widely performed in human or veterinary
medicine.
Serum bactericidal testing (SBT) is performed in the same
manner as the MBC test, but uses the serum of a patient that has
been treated with an antimicrobial. Serum is usually ­collected
at expected peak and trough serum concentrations. SBC can be
used to determine whether a patient has been adequately dosed,
and to ensure that the antimicrobial is having the desired effect
on the organism. Some data are available to support specific
peak and trough SBT results required for the optimal treatment
of human endocarditis, osteomyelitis, and cancer chemotherapy
patients.17-20 The SBT has not been widely used or evaluated for
infections in dogs and cats and remains controversial in human
medicine.
Mutant Prevention Concentration
The mutant prevention concentration (MPC) is the lowest anti-
microbial drug concentration required to block the growth of
the least susceptible bacterial cell in high density bacterial pop-
ulations.21 It is the MIC of the most resistant bacterial strain
in a mixed bacterial population. Concentrations of antimicro-
bial between the MIC and the MPC allow selective amplifica-
tion of resistant mutants (also known as the mutant selection
window).22 This range is considered to be a “danger zone”
for therapeutic drug concentrations. Minimizing the length of
time that the antimicrobial drug concentration remains in the
mutant selection window in vivo may reduce the likelihood for
resistance selection during treatment.22 The MPC cannot be
extrapolated from the MIC. It is estimated using the standard
agar dilution method used to estimate MIC, but with a larger
inoculum (≥109 instead of 105 organisms), so as to include
resistant subpopulations of bacteria. Administration of higher
doses of antimicrobial drugs that exceed the MPC increases
the chance of toxicity to the patient, but offsets the chance of
selection for resistant organisms, even though infection may
be cured with lower doses.22,23 Although first evaluated for
the fluoroquinolones, data is accumulating regarding the use-
fulness of the MPC for other drug classes. MPC estimations
are currently not routinely performed in veterinary diagnostic
laboratories.
SUGGESTED READINGS
Drlica K, Zhao X. Mutant selection window hypothesis updated. Clin
Infect Dis. 2007;44(5):681-688.
Turnidge JL, Paterson DL. Setting and revising antimicrobial suscepti-
bility breakpoints. Clin Microbiol Rev. 2007;20(3):391-408.
REFERENCES
1. Radaelli ST, Platt SR. Bacterial meningoencephalomyelitis in dogs:
a retrospective study of 23 cases (1990-1999). J Vet Intern Med.
2002;16(2):159-163.
2. Sturges BK, Dickinson PJ, Kortz GD, et  al. Clinical signs, mag-
netic resonance imaging features, and outcome after surgical and
medical treatment of otogenic intracranial infection in 11 cats and
4 dogs. J Vet Intern Med. 2006;20(3):648-656.
3. Cole LK, Kwochka KW, Kowalski JJ, et  al. Microbial flora and
antimicrobial susceptibility patterns of isolated pathogens from the
horizontal ear canal and middle ear in dogs with otitis media. J Am
Vet Med Assoc. 1998;212(4):534-538.
4. Tolar EL, Hendrix DV, Rohrback BW, et  al. Evaluation of
clinical characteristics and bacterial isolates in dogs with bac-
terial keratitis: 97 cases (1993-2003). J Am Vet Med Assoc.
2006;228(1):80-85.
5. Clinical and Laboratory Standards Institute. http://www.clsi.org:
Last accessed May 15, 2012.
6. Sykes JE, Kittleson MD, Pesavento PA, et al. Evaluation of the rela-
tionship between causative organisms and clinical characteristics of
infective endocarditis in dogs: 71 cases (1992-2005). J Am Vet Med
Assoc. 2006;228(11):1723-1734.
7. Thomson RB. Specimen collection, transport, and processing:
bacteriology. In: Murray PR, Barron EJ, Jorgensen JP, et  al. eds.
Manual of Clinical Microbiology. 9th ed. Washington, DC: ASM
Press; 2007:291-333.
8. Veir JK, Ruch-Gallie R, Spindel ME, et al. Prevalence of selected
infectious organisms and comparison of two anatomic sampling
sites in shelter cats with upper respiratory tract disease. J Feline
Med Surg. 2008;10(6):551-557.
9. Johnson LR, Drazenovich NL, Foley JE. A comparison of routine
culture with polymerase chain reaction technology for the detection
of Mycoplasma species in feline nasal samples. J Vet Diagn Invest.
2004;16(4):347-351.
10. Randolph JF, Moise NS, Scarlett JM, et al. Prevalence of mycoplas-
mal and ureaplasmal recovery from tracheobronchial lavages and
of mycoplasmal recovery from pharyngeal specimens in cats with
or without pulmonary disease. Am J Vet Res. 1993;54:897-900.
11. Tan RJS, Lim EW, Ishak B. Ecology of mycoplasmas in clinically
healthy cats. Aust Vet J. 1977;53:515-518.
12. Ling GV. Lower Urinary Tract Diseases of Dogs and Cats. St Louis:
Mosby; 1995:116.
13. Murray PR, Barron EJ, Jorgensen J, et al. eds. Manual of Clinical
Microbiology. 9th ed. Washington, DC: ASM Press; 2007.
14. Bizzini A, Greub G. Matrix-assisted laser desorption ionization
time-of-flight mass spectrometry, a revolution in clinical microbial
identification. Clin Microbiol Infect. 2010;16(11):1614-1619.
15. Turnidge JL, Paterson DL. Setting and revising antimicrobial sus-
ceptibility breakpoints. Clin Microbiol Rev. 2007;20(3):391-408.
16. The European Committee on Antimicrobial Susceptibility Testing—
EUCAST. http://www.eucast.org. Last accessed May 14, 2012.
17. Klastersky J, Daneau D, Swings G, et  al. Antibacterial activity
in serum and urine as a therapeutic guide in bacterial infections.
J Infect Dis. 1974;129(2):187-193.
18. Reller LB. The serum bactericidal test. Rev Infect Dis. 1986;
8(5):803-808.
19. Weinstein MP, Stratton CW, Ackley A, et al. Multicenter collaborative
evaluation of a standardized serum bactericidal test as a prognostic
indicator in infective endocarditis. Am J Med. 1985;78(2):262-269.
20. Weinstein MP, Stratton CE, Hawley HB, et  al. Multicenter col-
laborative evaluation of a standardized serum bactericidal test as a
predictor of therapeutic efficacy in acute and chronic osteomyelitis.
Am J Med. 1987;83(2):218-222.
21. Blondeau JM, Hansen G, Metzler K, et  al. The role of PK/PD
parameters to avoid selection and increase of resistance: mutant
prevention concentration. J Chemother. 2004;16(suppl 3):1-19.
22. Drlica K, Zhao X. Mutant selection window hypothesis updated.
Clin Infect Dis. 2007;44(5):681-688.
23. Zhao X, Drlica K. A unified anti-mutant dosing strategy. J Antimi-
crob Chemother. 2008;62(3):434-436.
CHAPTER 4

Isolation and Identification of Fungi

Jane E. Sykes and Shelley C. Rankin

w KEY POINTS
• F ungi often grow more slowly than bacteria and can have spe-
cific culture media requirements. Some fungi are dangerous to
culture because they produce airborne spores that may lead to
laboratory-acquired infections.
• The clinician should tell microbiology laboratory personnel if
a fungal infection is suspected. Providing a list of suspected
pathogens facilitates safe and successful culture of pathogenic
fungi. The animal’s clinical abnormalities, travel history, history
of immunosuppressive drug therapy, and the results of cytol-
ogy or histopathology should be taken into account.
• S pecimens should be collected and transported as for bacterial
culture. Refrigeration inhibits growth of some fungi.
• Because of increasing drug resistance among pathogenic fungi,
the demand for antifungal susceptibility testing has increased,
and for some fungal pathogens, culture and susceptibility
testing before beginning antifungal drug therapy may be
advantageous.

INTRODUCTION AND DEFINITIONS can exist in more than one asexual form, and therefore have more
than two names. For example, Pseudallescheria boydii (teleomorph)
Fungi are eukaryotic microorganisms that secrete enzymes in has two asexual forms, Graphium fruticola and Scedosporium apio-
order to digest organic material in the environment. The resul- spermum. Currently, there is a move by mycologists to identify only
tant nutrients are absorbed through a specialized fungal cell wall a single name for a fungal organism (‘one fungus = one name’).
(known as chemotrophic nutrition). A basic understanding of fun- Throughout this book, the name most commonly used in the lit-
gal terminology facilitates communications between clinicians, erature is used to refer to different fungal pathogens.
microbiologists, and pathologists. Fungal organisms have two
basic morphologic forms: Specimen Collection and Transport
1. Molds—molds exist as filaments known as hyphae, which may
be divided into cellular compartments (septate hyphae). A mass Isolation of fungal organisms from clinical specimens can be
of filaments is known as a mycelium. A granule formed by a com- facilitated by the use of special media. For many fungal spe-
pacted mat of fungal filaments is a eumycetoma (as opposed to cies, this may be hazardous to laboratory personnel. Submission
an actinomycotic mycetoma; see Chapter 42). of specimens for culture of dangerous organisms such as Blas-
2. Yeasts—these are single-celled organisms that reproduce by tomyces dermatitidis, Coccidioides immitis, and Histoplasma
budding. In some cases, buds do not detach from the parent cell,
but instead form a chain. Elongated chains of buds may resemble
hyphae. These are termed pseudohyphae (Figure 4-1).
Many pathogenic fungi exist as a mold in the environment,
but in tissues convert to a yeast form. These fungi are called
dimorphic fungi. An example of a dimorphic fungus is Blastomyces
dermatitidis.
Fungi reproduce by producing spores. Spores can be produced
following asexual or sexual reproduction. Asexual reproduction
(through mitosis) generates spores that are identical to the parent
cell. Sexual reproduction involves the fusion of haploid nuclei from
two hyphal structures, which is followed by meiosis. The asexual
stage of a fungus is known as an anamorph, whereas the sexual
stage is known as a teleomorph. Many fungi have two different spe-
cies names to reflect these stages. For example, the teleomorph
of Cryptococcus neoformans is known as Filobasidiella neoformans.
Formally, the teleomorph name is supposed to take precedence
and can be used to describe both stages.1 However, asexual repro-
duction predominates in most fungi, including during growth in FIGURE 4-1  Histopathology of the thyroid gland from a 13-year old female spayed
culture. Thus, certain fungi, such as C. neoformans, are more widely poodle mix with disseminated Candida albicans infection. The dog had been treated with
known by their anamorph name. In addition, teleomorphs of some prednisone, cyclosporin, and azathioprine for refractory immune-mediated hematologic
fungal organisms have not yet been described. Some organisms disease. Note intralesional pseudohyphae. H&E stain, 400× magnification.

29
30 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
capsulatum is discouraged if the diagnosis can be obtained using
other methods such as cytology, histopathology, and serologic
testing. These organisms exist as yeasts in tissues (at 37°C) that
are minimally hazardous to clinicians and laboratory person-
nel, but grow as hyphae at lower temperatures and on culture
medium, which generate spores that can be inhaled and cause
disease.
Specimens that are commonly submitted for fungal culture
include lower respiratory tract wash specimens, cerebrospinal
fluid, urine, other body fluids, skin, eye, hair, or biopsy speci-
mens from the nasal cavity, bone, or skin. Guidelines for speci-
men collection are the same as for bacterial culture (refer to
Chapter 3). The clinician should inform the laboratory what
fungal species are suspected, because different fungi have differ-
ent media preferences and growth conditions. As large a speci-
men as possible should be collected. Care should be taken to
avoid collection of contaminated specimens. Blood cultures may
be useful for dogs and cats with disseminated fungal disease
when lesions cannot be easily accessed. Detection of fungemia
can sometimes be achieved using blood culture systems for bac-
terial isolation. Lysis-centrifugation methods (Isolator tubes,
Wampole Laboratories) may be more sensitive for detection of
dimorphic and filamentous fungi in blood. Mycobacterial blood
culture media (such as MB/BacT, BioMérieux) may also offer
increased sensitivity for detection of fungemia.2
For dermatophyte culture, 10 to 12 hairs should be plucked
from the edge of the lesion using sterile forceps and submit-
ted without refrigeration in a clean dry paper envelope or ster-
ile glass or plastic tube. Paper envelopes are preferred to tubes
because tubes retain moisture that can promote overgrowth of
contaminants. Scrapings of crusts at the edges of the lesion can
also be submitted. Claw clippings can be collected after cleaning
them with 70% alcohol to remove contaminating bacteria and
fungi. Crumbling material from under the claw may be the best
specimen, or the entire claw can be submitted.
If transport requires longer than 2 hours, appropriate trans-
port media should be used or specimens should be refrigerated.
When rapid transport to the laboratory is possible, storage and
transport at room temperature is preferred. Fungal culture of
specimens that are more than 24 hours old is not recommended
because of increased likelihood of false-positive and false-nega-
tive results. The laboratory should be informed if the patient is
receiving antifungal drug treatment, which may interfere with
the ability to culture fungus from the specimen.
Specimens should be labeled with the patient’s name, patient
number, and source of specimen, as well as the date and time
of collection, and labeled, packaged, and transported accord-
ing to regional and global regulations for shipping of infectious
substances (see Chapter 1).3 Careful packaging and transport
is particularly important for specimens suspected to contain
fungal pathogens. If dangerous organisms might be present,
the laboratory should be warned in advance and the specimens
marked appropriately.
Diagnostic Methods
Microscopic Examination of Direct Smears
Examination of a direct smear before culture allows rapid par-
tial or complete identification of many fungi by the laboratory
(Table 4-1, Figure 4-2). This can help the clinician make initial
treatment decisions and guides media selection by the labora-
tory. Using Gram stain, yeasts generally stain gram positive
and molds tend to be gram negative.4 A potassium hydroxide
(KOH) preparation may be performed to examine a specimen
for dermatophyte hyphae and arthroconidia. The KOH clears
keratinaceous and cellular debris, but fungal elements are left
intact. If tissue granules are present, the laboratory will crush
them, examine them for hyphae, and the granules can also be
cultured. Fungal morphology may be altered in specimens from
dogs and cats receiving antifungal drug treatment (Figure 4-4).
Special stains used in the laboratory for fungal identification
include India ink, which is used to identify cryptococcal yeasts
in cerebrospinal fluid (see Figure 62-8), and calcofluor white,
which binds polysaccharides in the fungal cell wall and fluo-
resces when examined using fluorescence microscopy ­(Figure
4-5, A). Other stains, such as lactophenol cotton blue (see
­Figure 4-5, B), are also used to identify fungi after they have
been grown in culture.
In some situations, fungi are observed in tissues with histo-
pathology but a culture of the causative organism is not avail-
able, either because it has not been performed or because the
organism has failed to grow. The morphology and staining
characteristics of the organism on histopathology may also be
useful for preliminary identification or allow full identification
of the pathogen. Special stains used include Mayer’s mucicar-
mine stain, which stains the cryptococcal polysaccharide cap-
sule red; Gomori’s methenamine silver stain, which stains fungal
organisms black; and periodic acid–Schiff, which stains fungal
elements dark pink (Figure 4-6). If the identity of the fungus
remains in doubt, an additional specimen should be collected
for culture if possible, and the microbiology laboratory should
be made aware of the morphology of the organism and the dif-
ferential diagnoses. Complete identification of the organism
using culture is useful for proper treatment and prognostication.
Fungal Culture
Before culture, clinical specimens must be prepared for inocula-
tion. For example, body fluids are usually first centrifuged by
the laboratory to concentrate fungal organisms. The sediment is
then resuspended in a smaller volume of supernatant and inocu-
lated onto selective media. If biopsy specimens are submitted,
the tissues are finely sliced, ground, or minced, which releases
the organisms before they are inoculated onto the media.
Many types of media are available for isolation of fungi,
and no single medium is optimal for all organisms. Many
fungi grow on blood agar plates used for primary isolation
of bacteria. Special media used for initial (primary) isolation
of fungal organisms from tissues and body fluids are shown
in Table 4-2. Other media are available for differentiation of
fungal species (see Fungal Identification, later). Media that
contain antibacterial agents (usually chloramphenicol or gen-
tamicin) are used to culture specimens that might contain bac-
teria. Cycloheximide is added to dermatophyte test medium
to inhibit the growth of contaminant saprophytic mold spe-
cies. This allows selective isolation of dermatophytes such as
Microsporum canis.
Plates for fungal culture are sealed with an air-permeable
tape in order to prevent exposure of laboratory personnel to
potentially dangerous spores and to minimize plate contami-
nation with airborne fungal organisms. Plates that grow fila-
mentous fungi are opened only in a biological safety cabinet.
Laboratories prefer to use media in tubes or bottles to reduce
occupational exposure to aerosolized fungal elements. Fungal
cultures are incubated at room temperature (22°C to 25°C)
 CHAPTER 4  Isolation and Identification of Fungi 31

TABLE 4-1
Microscopic Descriptions of Some Common Fungal Pathogens of Dogs and Cats
Yeast or Geographic
Organism Hyphae Diameter (µm) Description Distribution
Blastomyces Yeast 8-15 Round with thick, often refractile cell Southeastern and south
dermatitidis wall and broad-based budding (see central United States,
Figure 60-8). upper Midwest region
Histoplasma Yeast 2-5 Oval to round, often found in clusters Ohio and Mississippi
capsulatum within macrophages (see Figure 61-7). river valleys, especially
in southern United
States. Also Central
America
Cryptococcus spp. Yeast 2-15 Round to oval, narrow-based bud- Worldwide, common in
ding, sometimes forming chains of Australia and western
organisms. Large capsule (see Figures North America
4-4 and 62-8). Poorly encapsulated
variants are rarely encountered.
Coccidioides spp. Spherule 10-200 Spherules vary in size and may contain Central California,
endospores (“pomegranate-like”). ­Arizona, Nevada,
Using cytology, they appear as empty Central and South
and collapsed, deeply basophilic America
structures (see Figure 63-8).
Sporothrix Yeast 2-6 Oval, round, or cigar-shaped, occasion- Temperate and tropi-
schenckii ally budding (see Figure 64-4). May cal zones worldwide.
be found intracellularly and extra­ Most common in
cellularly. Difficult to find in dogs South and Central
but may be abundant in cats. America
Malassezia spp. Yeasts and 3-8 (yeast), 5-10 Round to oval; when budding, have Worldwide
­pseudohyphae (pseudohyphae) the appearance of “footprints” (see
Figure 59-2).
Candida spp. Yeast and 3-4 (yeast), 5-10 Usually yeasts with a single bud Worldwide
­pseudohyphae (pseudohyphae) or pseudohyphae with multiple
­constrictions (see Figure 67-3).
Dermatophytes Hyphae 3-15 Septate hyaline hyphae, arthroconidia Worldwide
may be visible along hair shafts (see
Figure 58-8)
Aspergillus spp. Hyphae 4-6 Septate hyaline hyphae with uniform Worldwide
diameter that branch at 45° angles
(see Figure 4-2).
Scedosporium spp., Hyphae 3-12 Septate hyaline hyphae that tend to Worldwide
Paecilomyces spp. branch at wider angles than Aspergillus
spp. (45° to 90°) (see Figure 4-6).
Basidiobolus and Co- Hyphae 10-30 Wide, non- or poorly septate irregular Tropical and subtropical
nidiobolus (zygomy- hyaline hyphae, branching at right regions of the
cosis) angles, may be twisted or folded. Americas, Africa,
Poorly stained using hematoxylin southeast Asia, and
and eosin stain (see Figure 4-3). Australia
Phialophora, ­Exophiala, Hyphae 1.5-6 Pigmented hyphae, budding cells with Worldwide
­Cladophialophora, single septa and chains of swollen
Bipolaris (phaeohy- round cells (see Figure 68-5). May
phomycosis or dema- form granules.
tiaceous molds)
Pythium insidiosum Hyphae 4-9 Sparsely septate, hyaline hyphae with Tropical, subtropical,
90° branches, detectable using hema- and some temperate
toxylin and eosin stain regions of the world.
32 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

FIGURE 4-2  Direct smear of an aspirate from a mediastinal mass of a 6-­year-old
female spayed labrador retriever with disseminated Aspergillus deflectus infection. Note
hyphae with parallel walls that branch at 45° angles. Stain uptake by some hyphae is poor.
Wright’s stain, 1000× oil magnification. (Image courtesy Dr. William ­Vernau, University of
California, Davis.)
FIGURE 4-3  Histopathology showing severe pulmonary zygomycosis at necropsy
in an 11-year-old mixed-breed dog that had been diagnosed with diabetic ketoacidosis
before euthanasia. H&E stain, 100× magnification.

A
A

B
FIGURE 4-5  Special stains used for identification of fungal isolates following direct
B
smear preparation. A, Calcofluor white stain of a mass of Aspergillus fumigatus hyphae.
FIGURE 4-4  A, Cerebrospinal fluid cytology (Cytospin preparation) from a dog with 1000× oil magnification. B, Lactophenol cotton blue preparation of the mold phase of
newly diagnosed cryptococcosis. Note the thick capsule, which appears as a halo around Histoplasma capsulatum. 1000 × oil magnification.
the organism, and narrow-based budding. Wright-Giemsa stain, 1000× oil magnification.
B, Cytology of a lymph node aspirate from a cat that had been treated for cryptococco-
sis with antifungal drugs. The architecture of the organisms is distorted. Diff Quik stain, 
1000 × oil magnification.
 CHAPTER 4  Isolation and Identification of Fungi 33

A B
FIGURE 4-6  Histopathology of the kidney from a 2-year-old female spayed German shepherd with disseminated paecilomycosis. A, Periodic acid Schiff stain. The fungal hyphae stain
bright pink. B, Gomori’s methenamine silver stain, which stains the fungal hyphae black. 400× magnification.

TABLE 4-2
Special Medium Types Used to Culture Fungi That Infect Dogs and Cats
Type of Medium
Sabouraud dextrose agar
Brain-heart infusion agar
Potato dextrose agar or potato None
flake agar
Inhibitory mold agar
Dermatophyte test medium
Additives Indications
None Cultivation of all fungi.
None Cultivation of all fungi.
Cultivation of all fungi. Especially useful for opportu-
nistic molds. Induces sporulation.
Chloramphenicol, sometimes gentamicin Cultivation of all fungi when bacterial contamination
is suspected or possible.
Chloramphenicol, gentamicin, Dermatophyte isolation. Shows a color change when
cycloheximide dermatophytes grow on the medium.

or 30°C, a lower temperature than that used for routine cul-
ture of bacteria. In general, cultures should be examined daily
for 2 weeks and twice weekly for an additional 2 to 4 weeks,
because the growth of some organisms may be very slow. In
general, rapidly growing fungi grow in 1 to 3 days, those with
intermediate growth in 5 to 9 days, and slow growers take up
to 4 weeks. Yeast colonies are smooth and resemble bacterial
colonies, and molds grow in colonies that are dry, wrinkled, or
heaped ­(Figure 4-7).
fungi. A variety of broth or solid media are also used for fun-
gal identification. For example, Candida species can be differ-
entiated using Candida bromcresol green (BCG) agar, because
each species has a specific colonial morphology and color on
this medium. Test strips are commercially available that allow
multiple biochemical identification tests for yeasts to be per-
formed simultaneously (e.g., API 20C AUX Yeast Identification
Kit, bioMérieux) (see Figure 3-8), so identification can be made
within 1 to 2 days.

Fungal Identification Interpretation of Fungal Culture Results

After growth in culture, fungi are identified based on visual
characteristics such as colony morphology and color. Light
microscopy is useful to evaluate the microscopic morphology
of yeasts and to determine the presence of septate or nonseptate
hyphae and fruiting structures for molds (Table 4-3). Increas-
ingly, DNA sequence information has been used to identify
fungi (such as after PCR of D2 or ITS regions of the 23S rRNA
gene). Matrix-assisted laser desorption ionization time-of-flight
(MALDI-TOF) mass spectrometry holds considerable promise
for identification of fungi in the future (see Chapter 3).5 Molds
that produce pigment are known as dematiaceous fungi (see
Figure 4-7, B), and those that do not are known as hyaline
False-negative and false-positive results are very common with
fungal culture, and results must be interpreted in light of the
organism grown, the animal’s clinical condition, and the results
of cytology and histopathology.
Specimens That Test Negative
Negative test results can occur as a result of inadequate speci-
men size (such as when swabs or aspirates are submitted), when
there are inadequate organism numbers at the site of specimen
collection, when overgrowth with contaminating bacteria or
saprophytic fungi occurs, or when there is loss of organism
34 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

viability during specimen transportation. If the patient is receiv-

ing antifungal drugs but has not completely cleared the infec-
tion, culture may be negative.
False-negative test results also occur when the organism is
not inoculated onto the correct media or sufficient time is not
allowed for growth. It is critical that clinicians inform the lab-
oratory if a fungal infection is suspected, and what organism
might be involved. Poor laboratory quality control procedures
may also lead to false-negative test results. Use of an accredited
laboratory can help minimize such problems.

Specimens That Test Positive

The detection of fungal organisms within a specimen does not
always imply that the organism is causing the animal’s clinical
signs. Contamination by saprophytic fungi, especially Aspergil-
lus, is the most common cause of false-positive fungal cultures.
Organisms are more likely to be clinically significant when cyto-
A logic examination of a stained smear also demonstrates the pres-
ence of fungal organisms.

Antifungal Susceptibility Testing

B from a single patient allows the development of resistance to
FIGURE 4-7  A, Growth of Cryptococcus gattii yeasts on potato flake agar. B, Clado-
phialophora bantiana, a pigmented (dematiaceous) mold, isolated from canine brain tis-
sue on potato flake agar.
Increasing resistance to antifungal agents has been docu-
mented for a variety of fungal pathogens. This has led to
an increased demand for antifungal susceptibility testing by
clinicians. Fortunately, the reproducibility of antifungal sus-
ceptibility testing has improved greatly in recent years, and
breakpoints (see Chapter 3) have been established for treat-
ment of many fungal infections in human patients. Although
breakpoints are still lacking for veterinary patients, antifun-
gal susceptibility testing allows comparison of the activity of
two or more antifungal drugs, and serial testing of isolates
be monitored over time. Methods of susceptibility testing that
have been adapted for yeast and mold infections include broth
microdilution, the Etest, and disk diffusion (see Chapter 3).

TABLE 4-3
Classification of Fungi That Are Known to Infect Dogs and Cats
Phylum Class Example Characteristics
Zygomycota Zygomycetes Absidia, Rhizopus, Conidiobolus, Aseptate hyphae. In asexual reproduction, spores
Basidiobolus are abundantly produced in a closed sac called
a sporangium. Sexual reproduction results in a
single, large thick-walled zygospore.
Basidiomycota Basidiomycetes Cryptococcus, Malassezia Sexual reproduction leads to haploid production
of basidiospores on a basidium.
Ascomycota Archiascomycetes Pneumocystis Sexual reproduction leads to haploid production
Hemiascomycetes Candida spp. of ascospores in a sac-like structure (ascus).
Euascomycetes Histoplasma, Blastomyces, Organisms are more commonly identified based
­Microsporum, Trichophyton on characteristics of asexual reproduction.
Aspergillus spp.
Fusarium spp.
Pseudallescheria spp. (teleomorph
of Scedosporium spp.)
 CHAPTER 4  Isolation and Identification of Fungi 35

Common antifungal agents tested include amphotericin B,
itraconazole, fluconazole, voriconazole, posaconazole, caspo-
fungin, griseofulvin, and flucytosine (Figure 4-8). Limitations
of antifungal susceptibility testing include the slow growth
of fungal pathogens when compared to bacteria, and the fact
that fungi may grow as yeasts or molds depending on media
and incubation conditions. Partial inhibition of growth over
a wide range of antifungal drug dilutions may also occur
(a phenomenon known as trailing). Criteria have been set for
interpretation of MICs in the presence of trailing for differ-
ent antifungal drugs.6 MALDI-TOF mass spectrometry can be
used to determine antifungal drug susceptibility among fungal
pathogens and in future may overcome some of the difficulties
that currently exist.7

1 2 3 4 5 6 7 8 9 10 11 12
A POS AND AND AND AND AND AND AND AND AND AND AB
0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 0.12
B MF MF MF MF MF MF MF MF MF MF MF AB
0.008 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 0.25
C CAS CAS CAS CAS CAS CAS CAS CAS CAS CAS CAS AB
0.008 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 0.5
D FC FC FC FC FC FC FC FC FC FC FC AB
0.06 0.12 0.25 0.5 1 2 4 8 16 32 64 1
E PZ PZ PZ PZ PZ PZ PZ PZ PZ PZ PZ AB
0.008 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 2
F VOR VOR VOR VOR VOR VOR VOR VOR VOR VOR VOR AB
0.008 0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 4
G IZ IZ IZ IZ IZ IZ IZ IZ IZ IZ IZ AB
0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 8
H FZ FZ FZ FZ FZ FZ FZ FZ FZ FZ FZ FZ
0.12 0.25 0.5 1 2 4 8 16 32 64 128 256

POS, positive control; AND, anidulafungin; MF, micafungin; CAS, caspofungin; FC, 5-flucytosine; PZ,
posaconazole; VOR, voriconazole; IZ, itraconazole; FZ, fluconazole; AB, amphotericin B.
B
FIGURE 4-8  A, Minimum inhibitory concentration (MIC) assay for a Candida krusei isolate. Growth in the presence of the antifungal drug is characterized by formation of a pellet on
the bottom of the well and a change in the color of the medium from purple to pink. B, Schematic of the same microtiter plate showing antifungal drug dilutions. Organisms are defined
as susceptible, intermediate, or resistant based on MIC cutoffs (breakpoints or interpretive criteria) set by regulatory bodies such as the Clinical and Laboratory Standards Institute in the
United States. The report to the clinician would read susceptible to anidulafungin (0.12 µg/mL), susceptible to micafungin (0.25 µg/mL), susceptible to caspofungin (1 µg/mL), intermediate
susceptibility to flucytosine (8 µg/mL), susceptible to voriconazole (0.12 µg/mL), susceptible to itraconazole (0.25 µg/mL), and resistant to fluconazole (128 µg/mL). The MICs for posacon-
azole and amphotericin B were reported as 0.25 µg/mL and ≤0.12 µg/mL, but the organism was not classified as susceptible or resistant because interpretive criteria for this organism-drug
combination were not available.
36 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
REFERENCES
1. Pitt JI, Samson RA. Nomenclatural considerations in naming spe-
cies of Aspergillus and its teleomorphs. Stud Mycol. 2007;59:67-70.
2. Taniguchi T, Ogawa Y, Kasai D, et al. Three cases of fungemia in
HIV-infected patients diagnosed through the use of mycobacterial
blood culture bottles. Intern Med. 2010;49:2179-2183.
3. World Health Organization. A guide for shipping infectious
­substances. 2009. http://www.who.int/ihr/infectious_substances/en/
index. Last accessed May 11, 2012.
4. Shea YR. General approaches for direct detection of fungi. In: Ver-
salovic J, Carroll KC, Funke G, et al. eds. Manual of Clinical Micro-
biology. 10th ed. Washington, DC: ASM Press; 2011:1776-1792.
5. Iriart X, Lavergne RA, Fillaux J, et  al. Routine identifica-
tion of medical fungi by MALDI-TOF: performance of the new
VITEK MS using a new time-effective strategy. J Clin Microbiol.
2012;50(6):2107-2110.
6. Johnson EM, Espinel-Ingroff AV, Pfaller MA. Susceptibility test
methods: yeasts and filamentous fungi. In: Versalovic J, Carroll
KC, Funke G, et al. eds. Manual of Clinical Microbiology. 10th ed.
Washington, DC: ASM Press; 2011:2020-2037.
7. Marinach C, Alanio A, Palous M, et  al. MALDI-TOF MS-based
drug susceptibility testing of pathogens: the example of Candida
albicans and fluconazole. Proteomics. 2009;9:4627-4631.
CHAPTER 5
Nucleic Acid Detection Assays
Jane E. Sykes and Shelley C. Rankin
w KEY POINTS
• A  ssays for detection of nucleic acid such as polymerase chain
reaction assays are increasingly available commercially for
diagnosis of companion animal infectious diseases.
• Molecular diagnostic assays include those that involve amplifi-
cation (the copying of nucleic acid so that it can be more read-
ily detected) or probe-based assays that do not involve nucleic
acid amplification.
• Specimens for these assays should be collected using sterile
technique in order to minimize contamination.
INTRODUCTION
Assays that detect nucleic acid, also known as molecular diagnos-
tic assays, include probe-based assays such as in situ hybridiza-
tion, and methods that amplify DNA, such as the polymerase chain
reaction (PCR).
DNA or RNA probes are short, single-stranded nucleic acids
(usually <50 base pairs) that have a sequence that hybridizes to
(i.e., is complementary to) a specific segment of DNA from the tar-
get microorganism (Figure 5-1). For in situ hybridization, the probe
is labeled with a fluorescent or chemical tag so that the bound
probe can be detected. The probe is reacted with a tissue specimen
on a microscope slide in order to determine if the organism is pres-
ent in the specimen. For PCR, two specific short (approximately 20
bases) DNA sequences called primers, which are complementary
to the target microorganism’s DNA, are used in conjunction with
a machine called a thermocycler to amplify a specific segment of
DNA from just one copy to millions of copies that can be more eas-
ily detected. In situ PCR is a combination of these two techniques.1
Nucleic acid detection methods are ideally suited to detection
of organisms that are not easily found using cytology or histo-
pathology, are slow-growing, or are difficult to culture, as well as
when a rapid (<12 hours) diagnosis is required. Other applications
are shown in Box 5-1.
The use of nucleic acid–based assays for diagnosis has
increased dramatically in veterinary medicine over the past paraffin-embedded tissue specimens. Specimens that have been
10 years. Because of the exquisite sensitivity of some assays, espe-
cially those that involve DNA amplification, positive results may
reflect contamination that occurs in the laboratory. Contamina-
tion problems have decreased with increased automation and
the use of real-time PCR assays (see later). In human medicine,
proficiency testing programs have helped to overcome quality
assurance problems in laboratories that perform molecular diag-
nostic assays.2 Not all PCR assays for a specific pathogen are equal,
because the nucleic acid primers and probes and the equipment
used to run each assay may be different. So a PCR assay for Lepto-
spira performed in one laboratory may perform differently than an
assay used in another laboratory.
• I nterpretation of the results of molecular diagnostic tests
requires knowledge of the problems and pitfalls of each type
of assay. The results of testing at one laboratory may differ from
that at another because of variations in assay design.
• With increased automation, laboratory proficiency testing,
improved quality assurance, and standardization of reporting,
the availability and utility of molecular diagnostic tests for com-
panion animal infectious diseases will increase.
A plethora of nucleic acid detection methods have been devel-
oped since the advent of PCR, many of which are commercially
available for diagnosis of human infectious diseases but are not
yet being widely used in veterinary medicine. These are described
in detail elsewhere.1,3,4 The purpose of this chapter is to outline
(1) the indications for nucleic acid detection methods, (2) the
best ways to collect and transport specimens for these assays, (3)
the most commonly used methods in veterinary medicine, and
(4) guidelines for their interpretation.
Specimen Collection and Transport
The recommendations for specimen collection and transport pre-
sented here are based on the guidelines published by the Clinical
and Laboratory Standards Institute (CLSI).5 These guidelines are
updated on a regular basis and ensure that detection of nucleic
acids within clinical specimens is optimal. In some instances,
nucleic acid may be present and detectable in a specimen even
when the specimen has not been handled in a manner that is con-
sistent with the guidelines. Many laboratories publish their own
specific guidelines for specimen collection and transport. Speci-
mens should be properly packaged and labeled (see Chapter 3).
Nucleic Acid Probe Assays
In situ hybridization is generally performed on formalin-fixed,
archived for several years may still be adequate, even for detec-
tion of RNA, which is more labile than DNA.6,7 The optimum
specimen for fluorescence in situ hybridization (see later for dis-
cussion) is tissue that has been snap frozen in liquid nitrogen
after being embedded in optimal cutting temperature (OCT)
medium.5
Polymerase Chain Reaction
When collecting specimens for PCR, the timing of collection rela-
tive to the course of disease and the best specimen type must be
considered. For acute diseases, PCR is often most useful early in
the course of disease. For chronic diseases, timing is less critical.
37
38 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

A B
FIGURE 5-1  Fluorescence in situ hybridization (FISH). A, A nucleic acid probe that is complementary to a segment of leptospiral DNA is tagged with a fluorescent label and incubated
with a tissue section on a slide. B, Unbound probe is washed away, and the section is examined using fluorescence microscopy. (Leptospira FISH image courtesy Dr. Richard Goldstein.)

BOX 5-1
Applications of Nucleic Acid Based Assays

Application Examples
Detection of microbes that are not easily detected using Bartonella spp., viral pathogens
light microscopic examination of tissues or smears
Detection of microbes that are difficult or impossible to Hemotropic mycoplasmas, Bartonella spp., Mycobacterium
culture spp., Brucella spp.
Rapid detection of microbes to allow early institution of Influenza virus, hypervirulent FCV, rabies virus, Mycobacte-
infection control measures rium tuberculosis
Detection of microbes that are hazardous to culture in the H1N1 influenza virus, Francisella tularensis
laboratory
Detection of microbes in the face of inactivating substances Retrospective identification of an infecting microorganism in
such as antimicrobials or formalin formalin-fixed, paraffin-embedded tissues after histopathology
Detection of antimicrobial resistance genes Possession of mecA, the gene for methicillin resistance, by
staphylococci
Detection of novel pathogens using broad-spectrum PCR Bartonella henselae,* hemotropic mycoplasmas†
and sequencing
Monitoring organism load as a measure of disease progres- FIV viral load
sion or response to treatment
Identification of a cultured organism to the species level Nocardia and Actinomyces spp., many molds

FCV, Feline calicivirus.

*Anderson B, Sims K, Regnery R, et al. Detection of Rochalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J Clin
Microbiol 1994;32:942-948.
†Willi B, Boretti FS, Cattori V, et al. Identification, molecular characterization, and experimental transmission of a new hemoplasma isolate from a

cat with hemolytic anemia in Switzerland. J Clin Microbiol 2005;43:2581-2585.

Knowledge of organism shedding patterns can help to determine
the most appropriate timing of specimen collection. For example,
Leptospira organisms are primarily present in blood in the first
week of acute illness, after which they may be shed in the urine.8
Because contamination can occur outside the PCR labora-
tory as well as within the laboratory, collection of specimens
for nucleic acid testing should be performed aseptically. Gloves
should be worn, and disposable instruments should be used
(e.g., disposable blades, punch biopsy instruments).
In general, for detection of pathogens that contain DNA (i.e.,
all bacteria, fungi, some viruses), either fresh or frozen specimens
should be submitted. In general, DNA is stable in tissue for up
to 24 hours at 2°C to 8°C, at least 2 weeks at −20°C, and at
least 2 years at −20°C or below −70°C.5 Specimens held at room
temperature should be submitted immediately and reach the
laboratory within 24 hours. Specimens can also be refrigerated
and submitted on wet ice within 72 hours. Provided the target
of the assay is not an erythrocyte pathogen, erythrocytes should
be removed before storage if possible. Specimens should not be
stored in frost-free freezers, as these undergo repeated freeze-
thaw cycling, which can be associated with DNA degradation.9
Box 5-2 outlines recommendations based on specimen type.
 CHAPTER 5  Nucleic Acid Detection Assays 39

BOX 5-2
Guidelines for Collection and Transport of Specimens for Polymerase Chain Reaction Assays

Specimen Type Collection and Transport Recommendations

Whole blood and Use only EDTA or acid citrate dextrose (ACD) anticoagulant. Heparin inhibits PCR.* The use of hemolyzed
bone marrow specimens and frozen blood should be avoided if possible.
DNA: Submit specimens held at room temperature within 24 hours of collection. Specimens can be stored at
2°C to 8°C for 72 hours, or at −20°C or ≤−70°C for prolonged storage (removal of erythrocytes recommended).
RNA: Use stabilization solution or send immediately to the laboratory on wet ice. For storage, freeze
serum, plasma, or buffy coat specimens at or below −70°C.
Tissue specimens Optimal specimen is 1 to 2 g of fresh tissue. Rinse blood from tissue using sterile saline. Wrap in saline-
moistened gauze or paper. Avoid excessive dilution. Formalin-fixed, paraffin-embedded tissues can be used
when no other specimen is available but are not optimal.
DNA: Transport fresh tissue immediately to the laboratory on wet ice. Any storage should be at or below
−70°C, preferably after snap freezing in liquid nitrogen.
RNA: Use stabilizing solution, process within 1 hour of collection, or snap freeze in liquid nitrogen and
store at or below −70°C
Cerebrospinal DNA: Transport at 2°C to 8°C or freeze at −20°C or at or below −70°C
fluid RNA: Chill immediately on wet ice and deliver to the laboratory within 4 hours. Otherwise freeze ­immediately
(removal of contaminating erythrocytes recommended) and ship on dry ice.
Bronchoalveolar Transport at room temperature within 24 hours of collection. If a delay is anticipated, store for up to
lavage specimens 72 hours at 2°C to 8°C or freeze at or below −70°C.
Urine Minimize storage at room temperature. Storage and transport should be according to the specific assay performed.
Feces Use preserved specimen transport container, or transport under refrigeration at 2°C to 8°C.

Adapted from the CLSI guidelines MM13-A.

*Beutler E, Gelbart T, Kuhl W. Interference of heparin with the polymerase chain reaction. Biotechniques 1990;9:166.

RNA is highly susceptible to degradation during storage and
transport. For detection of RNA viruses, some laboratories pro-
vide an RNA stabilizing solution into which specimens are col-
lected to prevent RNA degradation. If stabilizing solution is not
readily available at the time of specimen collection, swabs and
body fluids should be immediately sent to the laboratory on wet
ice. Tissue should ideally be snap frozen at −70°C within half an
hour of collection and shipped on dry ice without being thawed in
the interim. RNA is stable for at least 2 years at or below −70°C.
Ribonucleases can continue to degrade RNA at −20°C.
For tissue specimens, the optimal amount is 1 to 2 g, although
more or less may be required depending on the cellularity of the
specimen.5 Tissues stored in formalin, especially for prolonged
periods, are suboptimal for PCR because the formalin cross-
links the DNA in the specimen. Tissues fixed briefly in formalin
and then paraffin-embedded are preferable to tissues stored in
formalin, because the formalin is removed during the embed-
ding process. Paraffin-embedded specimens should only be used
when no other specimens are available.5
Although PCR can be performed on feces, its sensitivity may
be lower than with other specimens because inhibitors of PCR
are particularly abundant in fecal material.10 The use of appro-
priate amplification controls by the laboratory facilitates detec-
tion of false negatives due to the presence of inhibitors.
Diagnostic Methods
Nucleic Acid Probes
A variety of nucleic acid probes are available commercially
as kits for detection of microorganisms that infect humans.
Some of these can also be used to detect the same pathogens
in specimens from dogs or cats. Probes are available for use in
the clinical microbiology laboratory to identify organisms that
have grown in culture (e.g., Gen-Probe Inc., San Diego), and
these probes are widely used in human clinical microbiology
laboratories.
Probe hybridization can be performed on a nitrocellulose
membrane (solid-phase hybridization); on formalin-fixed,
paraffin-embedded sections mounted on a microscope slide
(in situ hybridization); or in solution (liquid-phase hybridiza-
tion). For solid-phase hybridization, the probe is reacted with
microorganism DNA that has been immobilized on the mem-
brane. Unbound probe is washed away, and the bound probe
is detected using fluorescence, chemiluminescence, radioactivity,
or color development (in the same way that bound antibody or
antigen is detected in an ELISA or immunofluorescent antibody
assay). For in situ hybridization, formalin-fixed, paraffin-embed-
ded specimens are sectioned and mounted on a special slide. The
sections are deparaffinized, dried, and incubated with a solution
that contains the probe, so both the presence and the location
of the target pathogen within tissues can be identified. In situ
hybridization assays that include a fluorescent-labeled probe are
referred to as fluorescence in situ hybridization (FISH) assays
(see Figure 5-1).
Because liquid-phase hybridization occurs in solution,
unbound probe cannot be washed away. To overcome this
problem, a chemiluminescent acridinium ester label is attached
to the probe. A subsequent chemical hydrolysis step selectively
degrades only unbound probe. On addition of peroxides, the
intact (hybridized) probe then emits light.11
40 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

Although not yet widely used for veterinary applications,
peptide nucleic acid (PNA) probes are now increasingly avail-
able to detect target DNA. PNA probes are uncharged peptides
that mimic DNA and bind to complementary DNA sequences
just as a nucleic acid probe would.12,13 PNA probes lack the
net negative charge of nucleic acid probes; therefore, the elec-
trostatic repulsion that normally occurs when two negatively
charged DNA strands hybridize does not occur. The result is a
more stable and specific binding of the probe to its target, which
in turn can be associated with increased assay sensitivity and
specificity.
Branched DNA assays and hybrid capture assays are highly
sensitive hybridization methods that include steps to intensify
the signal generated from probe hybridization.3,4 They are not
yet widely used in veterinary medicine.
The Polymerase Chain Reaction
PCR allows the specific amplification of DNA sequences from
just one copy to millions of copies, which can be more readily
detected. The DNA from a clinical specimen is extracted using a
commercially available DNA extraction kit. A pair of primers,
roughly 20 nucleotides long, is then used to bracket a desired
DNA sequence, which is subsequently copied using a DNA
polymerase enzyme (Figure 5-2).
PCR assays are broadly divided into conventional PCR
assays and real-time PCR assays. Conventional PCR assay is
the simplest PCR method and although still widely for research
purposes, its use for diagnostic purposes is decreasing. In con-
ventional PCR, the PCR product is detected as a band on an
agarose gel after agarose gel electrophoresis. In real-time PCR,
a fluorescent signal is generated as PCR products accumulate,
allowing them to be detected in “real time.” A major advantage
of real-time PCR methods is that the tube in which the PCR
takes place does not need to be opened for detection of the PCR
product. This greatly reduces the likelihood of contamination
and false positive test results. Real-time PCR assays are ame-
nable to automation, and reactions are generally loaded onto
covered 96- or 384-well plates. This can be done robotically,
further reducing the possibility of contamination and manual
loading errors.
Regardless of the type of PCR assay, extracted DNA is added
to aliquots of a PCR mastermix. The mastermix is a solution

A C
B
D

FIGURE 5-2  Basic PCR assay. Nucleic acid is extracted from a clinical specimen through removal of contaminating proteins such as hemoglobin. Two primers are added that hybridize
at each end of a specific segment of a pathogen’s DNA. Also present in the reaction mixture are the enzyme Taq polymerase (dots) and free nucleotides (dNTPs) for new DNA. A thermocy-
cler is used to rapidly and repeatedly heat and cool the tubes through denaturation, annealing, and extension steps. Denaturation separates double-stranded DNA. Annealing occurs at a
specific temperature that causes the primers to bind only to their target sequences. When the reaction mixture is then heated to 72°C, the Taq enzyme uses the primers as initiation points
for DNA extension, and the target sequence is copied. The process is repeated 30 to 50 times, with logarithmic accumulation of the PCR product, so there are millions of copies of the target
pathogen’s DNA. These can then be detected using agarose gel electrophoresis (see Figure 5-3).
 CHAPTER 5  Nucleic Acid Detection Assays 41

that contains the two primers, dNTPs (G, T, A, and C) for mak-
ing new DNA, and a DNA polymerase. The DNA polymerase
used most commonly (Taq DNA polymerase) is a heat-stable
polymerase derived from Thermophilus aquaticus, a bacterium
that is adapted to live in hot springs. Typical PCR assay reaction
volumes are 10 to 50 µL.
The next step is performed in a thermocycler, a machine that
is programmed to heat and cool the tubes repeatedly, so that
the DNA can be amplified (see Figure 5-2). Initially the DNA
is heated to 95°C, which causes the paired strands of DNA to
denature. Next, the tubes are cooled to a specific annealing
temperature (usually around 50°C to 65°C), which allows the
primers bind to their respective sequences. Finally, the tubes are
heated to 72°C. This is the temperature at which the DNA poly-
merase adds dNTPs in the mastermix to the primers, extending
the primers as it copies the sequence of the template DNA. The
3-step cycle is repeated 30 to 50 times, which results in loga-
rithmic accumulation of a PCR product that is an exact copy
of the segment of DNA between the two primers. The amplified
segments of DNA are called amplicons.
Detection of the PCR Product: Conventional versus Real-time PCR
In conventional PCR, amplified DNA is loaded into wells in an
agarose gel then passed through the gel using horizontal electro-
phoresis, stained with a DNA stain, and visualized using ultra-
violet transillumination. The longer the PCR product, the more
slowly it moves through the gel during electrophoresis. When
a series of molecular weight markers are run alongside the end
product of PCR, the size of the PCR product can be estimated
(Figure 5-3). The true identity of the product can then be con-
firmed using sequencing or by transferring it to a membrane
and performing DNA hybridization with a specific probe. The
process of loading the PCR product in agarose wells requires
that the PCR tubes be opened, which can lead to aerosoliza-
tion of PCR amplicons. These amplicons can then contaminate
reagents, equipment, and tubes that are used for running sub-
sequent PCR assays. Unless proper controls are included, false
positives resulting from such contamination may go unidenti-
fied and lead to erroneous reporting of results.
Two major types of real-time PCR assays are in use in veteri-
nary medicine: fluorescent probe–based assays and SYBR Green
PCR assays.
Fluorescent probe real-time PCR assays incorporate one or
more specific DNA probes into the PCR mastermix, in addition
to the two primers used in conventional PCR. The probe(s) are
tagged with a dye that emits fluorescent light. Inclusion of a
probe adds specificity to the test, because it is less likely that
both primers and the probe will bind to DNA other than that of
the target organism. Three types of probes may be used:
1. TaqMan (5′ nuclease) probes. Many veterinary laborato-
ries that offer probe-based PCR assays use TaqMan probes.
These are tagged with a fluorescent reporter dye at one end,
and a quencher dye at the other end of the probe. The close
proximity of the quencher dye to the reporter dye inhibits
the reporter dye from emitting fluorescence. When the Taq
DNA polymerase copies the section of DNA between the
two primers, it encounters the probe and chops it into pieces,
because it has inherent exonuclease activity. This causes the
reporter dye to separate from the quencher dye. The reporter
dye then emits fluorescence, which can be measured using a
spectrophotometer (Figure 5-4, A).
2. Molecular beacons. A molecular beacon is a probe with fluo-
rescent reporter and quencher dyes at each end, but when
not bound to the target sequence, it forms a hairpin loop
that brings each dye in close proximity and stops the reporter
dye from emitting fluorescence. When the probe anneals, the
dyes separate, and the reporter dye emits fluorescence (see
Figure 5-4, B).
3. FRET probes. FRET (fluorescent resonance energy transfer)
probe assays use two probes that are expected to lie next
to one another on the target sequence. One of the probes is
tagged at the 3′ end with a green fluorescent dye, the other
at the 5′ end with a red fluorescent dye. When the two dyes
come together during annealing, the green fluorescent dye
transfers its energy to the red probe, causing it to emit flu-
orescence, which can then be detected (see Figure 5-4, C).
Currently there are only rare reports of the use of this tech-
nology for diagnosis in veterinary medicine.

A B
FIGURE 5-3  A, Detection of PCR products by agarose gel electrophoresis. At the end of the PCR process, the reaction mixture that results is loaded into wells at the top of an agarose
gel. An electric current is applied across the gel, and because DNA is negatively charged, any amplified DNA in the reaction mixture migrates through the gel toward the positive terminal.
The longer the DNA sequence that has been amplified, the more slowly it moves through the gel. Simultaneous electrophoresis of a molecular weight standard in an adjacent lane (left
lane) allows estimation of the size of any PCR products amplified. The molecular weight standard contains DNA fragments of known size. After electrophoresis, the gel is stained with a DNA
stain. If the target pathogen’s DNA was successfully amplified, it appears as a band of an expected size (in this case, 485 base pairs [bp] long). Bands that are the wrong size (“aberrant PCR
products”) may represent nonspecific amplification of DNA from a different microorganism or host DNA (middle lane). B, Photograph of PCR products in an agarose gel after agarose gel
electrophoresis and staining. DNA bands amplified from Chlamydia felis, feline calicivirus (FCV), and feline herpesvirus-1 (FHV-1) are shown. The primers were designed to amplify different-
length products from each pathogen so that if multiple pathogens were present in a single specimen, they could be distinguished from one another.
42 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases

A B

C
FIGURE 5-4  Probe-based real-time PCR assays. A, TaqMan probe PCR system. In addition to the forward and reverse primers used in conventional PCR, an additional short probe is used
that binds specifically to the DNA of the pathogen of interest (step one). Emission of fluorescence by a reporter dye located at one end of the probe is quenched by another dye at the other
end of the probe. When the Taq enzyme extends the primers, an inherent exonuclease activity of the enzyme results in the digestion of the probe. The quencher and reporter dyes separate,
causing the quencher dye to emit fluorescence. Other probe-based real-time PCR assays include molecular beacon assays (B) and fluorescent resonance energy transfer probe assays (C).

SYBR Green Real-time PCR assays use a double-stranded DNA
binding dye (SYBR Green) that fluoresces when bound to double-­
stranded DNA as it forms during PCR amplification. Because
there is no probe, carefully designed primers are required to
ensure high assay specificity for the organism of interest.
The amount of fluorescence emitted during real-time PCR is
proportional to the number of copies of template DNA in the
specimen, so real-time PCR assays can be quantitative. A plot of
PCR cycle number against fluorescence is generated by an attached
computer and interpreted by the laboratory technician (Figure
5-5). The number of cycles that elapse before fluorescence is
detectable above a certain threshold value is the CT (cycle thresh-
old) value. Most laboratories run approximately 40 cycles, and
so specimens that generate CT values less than 40 are considered
positive. Said another way, if PCR proceeds to 40 cycles with-
out emission of fluorescence (CT = 40), then the target DNA is
not detectable in the specimen, and the result is reported as nega-
tive. The lower the CT value, the more target DNA (= organism
load) is present in the sample, because fewer cycles elapse before
fluorescence is detected. Because specimen size can vary, the CT
value may be normalized to the number of host cells present in
the sample. Knowledge of organism load may have prognostic
value for some infections, can allow infection to be monitored
over time with treatment, and may be useful to differentiate low-
level colonization from active infection causing clinical signs (see
Interpretation of Results, later). At the time of writing, organism
loads are variably reported by veterinary diagnostic laboratories
performing PCR assays.
 CHAPTER 5  Nucleic Acid Detection Assays 43

FIGURE 5-5  Plot that shows the emission of fluorescence during PCR product formation over time in real-time PCR as the number of cycles of denaturation, annealing, and extension
increases from 0 to 40 (x-axis). The cycle threshold (CT) value is the number of PCR cycles required for the fluorescent signal (y-axis) to rise above a threshold value for fluorescence (threshold
line). The more copies of target pathogen DNA that are present in a clinical specimen (which correlates with the number of organisms present), the earlier fluorescence is emitted and the
lower the CT value. Thus, for example, if only one organism is present in a submitted specimen, the CT value for this particular PCR assay is 32, but if 10,000 organisms are present, the CT
value is around 19.

FIGURE 5-6  Nested PCR. After the first round of PCR amplification, a second set of internal primers (green) is used in yet another round of PCR amplification, which generates a second,
shorter PCR product.

Rapid thermocyclers are available in several different for-
mats. Special carousel thermocyclers have been developed that
rapidly heat and cool the reactions, which are placed in specially
designed glass capillaries. As a result, PCR assays can be com-
pleted in 45 minutes, instead of the usual 4 to 5 hours.14
Polymerase Chain Reaction Variations
Variations on the basic PCR assay include reverse-transcriptase
PCR, nested PCR, and multiplex PCR.
Reverse-transcriptase PCR is used for RNA detection. After
extraction of RNA from the clinical specimen using a com-
mercial kit, the enzyme reverse transcriptase is added to the
RNA, together with dNTPs that will be used to make DNA.
The starting point for reverse transcription can be a single
complementary DNA primer that binds to the RNA, or alterna-
tively, random hexamers, which are a mixture of six-nucleotide
primers that represent all possible combinations of dNTPs. The
reverse transcriptase then uses the primers to make a DNA copy
of the RNA (known as cDNA), which is subsequently subjected
to PCR amplification.
For nested PCR, a small amount of amplified PCR product is
subjected to a second round of conventional PCR using another
set of internal primers (Figure 5-6). Having an additional set
of primers increases the specificity of the PCR assay and also
greatly increases its sensitivity.15 Unfortunately, the increased
sensitivity means that false positives due to contamination can
be extremely problematic with this method, and it has lost pop-
ularity for diagnostic purposes.
44 SECTION 1  Laboratory Diagnosis of Canine and Feline Infectious Diseases
Multiplex PCR assays use multiple sets of primers in the same
reaction tube to simultaneously detect multiple organisms (or mul-
tiple genes from the same organism). Duplex PCR assays can detect
two organisms, and triplex PCR assays can detect three organisms.
The potential downside of multiplex reactions is that if both organ-
isms are present, and one organism is present in relative abun-
dance, amplification of this organism can consume reagents, which
reduces the sensitivity of the assay for the other target organism.
Verification of Polymerase Chain Reaction Assay Performance
Before being used for diagnostic purposes, PCR assays must be
shown to be sensitive, specific, reproducible, and efficient.
For PCR, the limit of detection is the lowest number of
copies of the target DNA or RNA sequence that the assay can
detect. Serial dilutions of known amounts of the template DNA
are tested using the PCR assay of interest, until template can no
longer be detected.
From a diagnostic standpoint, a PCR assay may lack sensi-
tivity (many false negatives) if the assay fails to detect all pos-
sible strains of a target organism. This can occur if the probe or
primers are strain, rather than species-specific, and many strains
of the target organism exist with variable sequences (as is the
case with RNA pathogens such as feline calicivirus). As part of
the verification process, the assay should be shown to detect as
many strains as possible.
A PCR assay has poor analytical specificity if false positives
occur as a result of contamination or because of manual loading
error. Specificity may also be poor if the primers bind to and ini-
tiate amplification of DNA from unrelated organisms or the host
animal (i.e., cat, dog, human DNA). Specificity is tested during
the verification process by showing that the PCR assay fails to
amplify DNA from specimens that contain organisms that are
genetically related to the target organism, as well as organisms
that may be expected to be contaminating normal flora.
The results of PCR assays must also be repeatable. Repeatabil-
ity is determined for real-time PCR assays by determining whether
the CT value varies significantly for the same specimen when
tested multiple times. Usually intraassay (within the same run)
and interassay (within different runs) variations are measured.
A real-time PCR assay that has an efficiency of 100% doubles
the number of copies of template in every PCR cycle. Efficiencies
below 90% are unacceptable for quantitative assays, and ideally
efficiency should be greater than 95%. The efficiency is deter-
mined by testing serial dilutions of a target DNA standard and
by creating a chart of copy number versus CT value. Ideally, this
is compared with a similar chart using dilutions of the standard
DNA spiked into a clinical specimen, to show that inhibitors
within the specimen will not affect efficiency.
Prevention of Contamination and Quality Control
Prevention of PCR contamination relies on good laboratory
practices, some of which are outlined in Box 5-3. Contamination
can be difficult to eradicate once it occurs, so prevention of con-
tamination is critical. Laboratories should include negative con-
trols with every run, and a positive control should be included
to ensure that the assay is working, preferably one that is put
through the nucleic acid extraction process. The positive con-
trol should contain a low concentration of DNA, as abundant
DNA in a positive control sample may be a source of contamina-
tion. Many laboratories run two separate negative controls. One
of the negative controls contains mastermix and nuclease-free
water in place of specimen DNA. The other consists of water or
a known negative specimen that is placed through the extraction
BOX 5-3
Methods That May Be Used to Minimize Contamination
in Diagnostic Laboratories That Perform Nucleic Acid
Amplification Assay
Separation of rooms for DNA extraction, PCR, and PCR
product detection
Robotic DNA extraction
Automated detection of PCR products (real-time PCR)
Opening one specimen tube at a time
Use of screw-top caps rather than snap-top tubes to pre-
vent splashing
Use of filter-plugged pipette tips
Use of negative extraction controls and negative template
controls in every run
Routine monitoring for contamination using wipes
Standard cleanup protocol that inactivates nucleic acids
Use of positive control samples of low concentration
Use of dUTP and uracil-N-glycosylase in the PCR
mastermix
process to ensure that contamination does not occur during
extraction (negative extraction control). When 96- or 384-well
plate formats are used, negative control wells are interspersed
among the test wells to monitor for plate contamination.
In order to prevent contamination, some laboratories use
deoxyuracil triphosphate (dUTP) instead of deoxythymidine
triphosphate (dTTP) in the PCR mastermix. The PCR products
therefore contain uracil (U) in place of thymidine (T). The enzyme
uracil-N-glycosylase (UNG) is also added to the mix. This enzyme
destroys DNA amplicons that contain deoxyuracil residues, and
eliminates any contaminating DNA amplicons from previous
reactions before PCR amplification begins. When the specimen
is heated in the initial denaturation step, the UNG is destroyed.
The presence of substances that inhibit the PCR assay can
be detected using a housekeeping gene control PCR assay. This
is performed on a separate aliquot of the specimen at the same
time as the assay for the target pathogen. Housekeeping genes
are host (dog or cat) genes that are expected to be present in the
specimen. Examples of commonly used housekeeping gene PCR
assays are those for canine or feline glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), histone, or 18S DNA. A negative
housekeeping gene PCR assay result (or a high CT value for the
assay, which is equivalent to a weak positive result) suggests
that inhibitors may be present in the specimen, or that the DNA
in the submitted specimen may have degraded during storage or
transport. Some PCR assays use an internal housekeeping gene
control, in which a housekeeping gene PCR is multiplexed with
the PCR assay for the target microorganism.
Interpretation of Results
Because some PCR assays can detect as few as one or two organ-
isms, and both viable and nonviable organisms are detected, it
may be difficult to determine the significance of a positive PCR
test result in light of an animal’s disease. An understanding of
the pathogenesis of infectious diseases and organism shedding
patterns is crucial in interpreting positive PCR results. Correct
interpretation of the results relies on knowledge of the methods
used by a laboratory and their advantages and disadvantages.

BOX 5-4
Factors That Should Be Considered When Interpreting
Nucleic Acid Amplification Assay Results
Negative Test Result
Organism not present
Too few organisms present
Insufficient specimen size
Wrong specimen type
Improper timing of specimen collection
Patient receiving antimicrobial therapy
Specimen degradation during storage or transport
Assay reagent failure
Assay inhibitors present or extraction failure
Reagents consumed in a multiplex assay
Assay does not detect all strains
Positive Test Result
Viable or nonviable organisms present and causing disease
Viable or nonviable organisms present but not causing
disease
Vaccine organisms present
Contamination
Loading errors
Assay cross-reaction with other pathogens or host DNA in
the sample
Specimens That Test Negative
Factors that lead to negative test results are shown in Box 5-4.
An example of the use of the wrong specimen type would be sub-
mission of blood for a Borrelia burgdorferi PCR test, when B.
burgdorferi is found predominantly in connective tissue.16 Assay
inhibition and specimen degradation can be identified by interpre-
tation of housekeeping gene control assay results, which should
be performed by the laboratory. Assay reagent failure is identified
by the laboratory when the positive control tests negative.
Specimens That Test Positive
Factors that must be considered when interpreting positive test
results are shown in Box 5-4. The most common reason for false-
positive test results for assays that involve DNA amplification is
contamination. Loading errors, in which specimens are inadver-
tently loaded into the wrong wells on a microtiter plate, may also
lead to false-positive results and are reduced with the use of auto-
mated methods. Because of the exquisite sensitivity of PCR, it may
be difficult to determine the significance of a true positive result.
The organism may be part of the normal flora, carried subclinically
(e.g., latent feline herpesvirus-1 in a nasal biopsy), or simply colo-
nizing the anatomic site sampled. Several U.S. veterinary laborato-
ries offer PCR “panels” for diagnostic purposes. Individual PCR
assays for several different pathogens are performed on nucleic
acid extracted from a single specimen. This high-throughput panel
format allows assays to be offered at a substantial discount over
the cost of multiple individual assays. This type of testing can result
in unexpected positive test results, such as a positive result for the
Clostridium perfringens toxin gene in a cat with diarrhea that is
suspected to have trichomoniasis. As with any laboratory test,
positive results need to be interpreted in light of the clinical history,
clinical signs, and the pathogenesis of the disease in question.
CHAPTER 5  Nucleic Acid Detection Assays 45
A positive result can occur if an attenuated live vaccine for
the target organism was recently administered. Because PCR
detects both viable and nonviable organisms, PCR can be posi-
tive for days after antimicrobials have been administered, when
culture is negative.17-19 The duration of PCR positivity after
treatment depends on the specimen type tested and the target
pathogen.
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1. Komminoth P, Werner M. Target and signal amplification:
approaches to increase the sensitivity of in situ hybridization. His-
tochem Cell Biol. 1997;108:325-333.
2. Quality Control for Molecular Diagnostics. 2012. http://www.qcmd.
org/. Last accessed May 11, 2012.
3. Andras SC, Power JB, Cocking EC, et al. Strategies for signal ampli-
fication in nucleic acid detection. Mol Biotechnol. 2001;19:29-44.
4. Nolte FS, Caliendo AM. Molecular microbiology. In: Versalovic J,
Carroll KC, Funke G, et al. eds. Manual of Clinical Microbiology.
10th ed. Washington, DC: ASM Press; 2011:27-59.
5. Clinical and Laboratory Standards Institute. Collection, transport,
preparation, and storage of specimens for molecular methods;
approved guideline. MM13-A. 2005;25:7-18.
6. Illig R, Fritsch H, Schwarzer C. Breaking the seals: efficient mRNA
detection from human archival paraffin-embedded tissue. RNA.
2009;15:1588-1596.
7. Tan LH, Do E, Chong SM, et al. Detection of ALK gene rearrange-
ments in formalin-fixed, paraffin-embedded tissue using a fluorescence
in situ hybridization (FISH) probe: a search for optimum conditions of
tissue archiving and preparation for FISH. Mol Diagn. 2003;7:27-33.
8. Greenlee JJ, Bolin CA, Alt DP, et  al. Clinical and pathologic
comparison of acute leptospirosis in dogs caused by two strains
of Leptospira kirschneri serovar grippotyphosa. Am J Vet Res.
2004;65:1100-1107.
9. Lahiri DK, Schnabel B. DNA isolation by a rapid method from
human blood samples: effects of MgCl2, EDTA, storage time, and
temperature on DNA yield and quality. Biochem Genet. 1993;31:
321-328.
10. Oikarinen S, Tauriainen S, Viskari H, et al. PCR inhibition in stool
samples in relation to age of infants. J Clin Virol. 2009;44:211-214.
11. Arnold Jr LJ, Hammond PW, Wiese WA, et  al. Assay for-
mats involving acridinium-ester-labeled DNA probes. Clin Chem.
1989;35:1588-1594.
12. Padilla E, Manterola JM, Rasmussen OF, et  al. Evaluation of a
fluorescence hybridisation assay using peptide nucleic acid probes
for identification and differentiation of tuberculous and non-­
tuberculous mycobacteria in liquid cultures. Eur J Clin Microbiol
Infect Dis. 2000;19:140-145.
13. Zhang N, Appella DH. Advantages of peptide nucleic acids as diag-
nostic platforms for detection of nucleic acids in resource-limited
settings. J Infect Dis. 2010;201(suppl 1):S42-S45.
14. Cockerill 3rd FR. Application of rapid-cycle real-time polymerase
chain reaction for diagnostic testing in the clinical microbiology
laboratory. Arch Pathol Lab Med. 2003;127:1112-1120.
15. Lu JJ, Chen CH, Bartlett MS, et  al. Comparison of six different
PCR methods for detection of Pneumocystis carinii. J Clin Microbiol.
1995;33:2785-2788.
16. Aguero-Rosenfeld ME, Wang G, Schwartz I, et  al. Diagnosis of
Lyme borreliosis. Clin Microbiol Rev. 2005;18:484-509.
17. Bockenstedt LK, Mao J, Hodzic E, et al. Detection of attenuated,
noninfectious spirochetes in Borrelia burgdorferi-infected mice
after antibiotic treatment. J Infect Dis. 2002;186:1430-1437.
18. Braddock JA, Tasker S, Malik R. The use of real-time PCR in the
diagnosis and monitoring of Mycoplasma haemofelis copy number in a
naturally infected cat. J Feline Med Surg. 2004;6:161-165.
19. Sykes JE, Studdert VP, Browning GF. Comparison of the poly-
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 SECTION 2
Antiinfective Therapy
Jane E. Sykes and Mark G. Papich
CHAPTER 6
Principles of Antiinfective Therapy
Jane E. Sykes and Mark G. Papich
w KEY POINTS
• T he use of antimicrobial drugs (AMDs) can lead to selection of
resistant bacterial populations, within the targeted bacterial
population and also other commensal bacteria.
• Bacterial mechanisms of drug resistance include production of
enzymes that inactivate AMDs, drug exclusion through mem-
brane alterations and efflux pumps, and modification of the site
of action of the AMD.
• Strategies that minimize selection for drug resistance include
(1) confirmation of the presence of infection before AMD treat-
ment is commenced, (2) proper identification of the infecting
INTRODUCTION
Antibiotics have been considered one of the greatest inventions
of the 20th century. The modern era of antimicrobial drug (AMD)
treatment began with the discovery of sulfonamides in 1935.1 In
the 1940s, the therapeutic value of penicillin and streptomycin
was discovered, and by 1950, the “golden age” of AMD therapy was
underway. With the increasing use of AMDs, there has been a transi-
tion to an era of widespread antimicrobial resistance among impor-
tant veterinary and human pathogens. This has been compounded
by a declining rate of development of new classes of AMDs.
Antimicrobial resistance genes have existed in microbes long
before antimicrobial agents were used in therapy. Microbe popula-
tions generally consist of a mixture of genetically susceptible and
resistant organisms. The use of AMDs exerts pressure that favors
selection of the resistant microbes (“survival of the fittest”), within
both the targeted bacterial population and other commensal bac-
teria (which have the potential to become pathogenic when host
defenses are impaired). The chance that resistant bacterial popula-
tions will emerge depends on the ability of the population to rapidly
acquire mutations, the ability of host defenses to eliminate resistant
bacteria, and the AMD concentrations at the site of infection.
Guidelines for prudent antimicrobial use have been published
by several professional veterinary organizations in order to reduce
selection for resistant bacterial pathogens.2-6 Individual hospitals are
46
organism, (3) selection of agents with activity specific to the patho-
gen present, (4) administration of an adequate dose of AMD, and
(5) administration of AMD for the proper duration of time.
• Selection of an appropriate AMD should be based on the activ-
ity of the agent against the suspected pathogen; knowledge
of drug pharmacokinetics, which includes bioavailability and
tissue penetration; consideration of host factors such as con-
current illness, medications, or immunosuppression; and knowl-
edge of drug pharmacodynamics, that is, whether the drug
exhibits concentration- or time-dependent antibacterial effects.
encouraged to develop antimicrobial guidelines based on evalua-
tion of their local prevalence of AMD resistance. Strategies that mini-
mize selection for antimicrobial resistance include documentation
of the presence of infection; proper identification of the infecting
organism; and the use of agents that are as specific for the pathogen
as possible, at the proper dose and for the proper duration of time.
These strategies are described in more detail in this chapter.
Identification of the Infecting Organism
The detection of fever or leukocytosis does not imply bacte-
rial infection and the need for antibiotic treatment, especially
given the relatively high prevalence of sterile inflammatory and
immune-mediated diseases in dogs and viral infections in cats.
Selection of the most appropriate AMD relies on knowledge of
(1) the presence of an infectious agent, (2) the type of infectious
agent present, and (3) the susceptibility of the agent to different
AMDs. In some cases, treatment with antibiotics may not be
necessary even though the cause of disease is bacterial. Some
infections undergo spontaneous cures without the aid of an
antibiotic. For example, a cat bite abscess can often be drained
and resolved without the need to administer an antibiotic (see
Chapter 57).
In some situations, collection of a specimen for cytology or
histopathology permits a diagnosis of infection to be established.

The identity of some infectious agents, such as rickettsial and
fungal pathogens, may be presumptively identified to the genus
or species level based on their morphology. If skin cytology from
a dog reveals cocci (a Gram stain is not necessary to confirm the
presence of gram-positive cocci), then the presence of Staphy-
lococcus pseudintermedius is highly probable. If, on the other
hand, cytology from an ear swab reveals rods, the possibility of
Pseudomonas aeruginosa must be considered. In either case, this
knowledge is valuable for selecting initial antibacterial therapy.
The use of molecular diagnostic techniques, such as PCR assays,
can also allow rapid identification of an infecting organism,
which can help to guide antimicrobial drug selection. For exam-
ple, identification of a vector-borne pathogen (e.g., Rickettsia
spp., Ehrlichia canis) is helpful because doxycycline is usually the
first drug of choice for the majority of these pathogens. Unfortu-
nately, unless susceptibility to a particular AMD is predictable,
these techniques do not typically provide information about
AMD susceptibility. Given the emergence of widespread AMD
resistance, especially among bacteria and some fungi, collection
of specimens for culture and susceptibility before treatment with
AMDs is commenced has become more and more important.
Although there is an initial cost to the client for culture and sus-
ceptibility testing, long-term costs to the client may be reduced
considerably because of decreased prescription of unnecessary or
inappropriate AMDs, improved likelihood of rapid resolution
of the underlying disorder with early diagnosis and appropri-
ate treatment, and reduced need for more expensive, second- or
third-line AMDs. Administration of AMDs without identifica-
tion of the infectious agent can also decrease subsequent chances
of successful culture and proper diagnosis if the disorder does
not resolve.
In animals with life-threatening infections, institution of
AMD treatment is necessary before the results of culture and sus-
ceptibility become available. The choice of treatment at this stage
can be based on cytology and knowledge of the type of infections
that most commonly occur at the anatomic site involved (also
known as bacteriologic statistics). Because of the life-threatening
nature of these infections, the practice of “de-escalation” (as
opposed to “escalation”) is employed. This is the practice of ini-
tially selecting a highly active agent that has a high probability of
successful elimination of the pathogen; then once the infectious
agent has been identified, the drug spectrum should be changed
or narrowed according to culture and susceptibility results. If
the infection is life threatening, highly active agents such as an
aminoglycoside or fluoroquinolone with or without a β-lactam
can be selected initially. Then, if culture and susceptibility show
susceptibility to β-lactams, treatment should be continued with
these agents alone.
Classification of Antimicrobial Drugs
Antimicrobial drugs may be classified based on their struc-
ture (e.g., β-lactams, fluoroquinolones), spectrum of activity
(gram-positive versus gram-negative bacteria), mechanism
of action (Table 6-1, Figure 6-1), whether they are bacterio-
static (inhibit or slow growth) or bactericidal drugs, and their
pharmacodynamic properties (see Pharmacodynamics of Anti-
microbial Drugs, later). Bactericidal drugs interfere with cell
wall or nucleic acid synthesis, whereas bacteriostatic drugs
inhibit protein synthesis or cause changes in bacterial physiol-
ogy. When a bacteriostatic drug is removed, in the absence of
host defense mechanisms, organism growth resumes, and any
CHAPTER 6  Principles of Antiinfective Therapy 47
decline in bacterial numbers that occurs when the drug con-
centration drops below the minimum inhibitory concentration
(MIC) reflects clearance by the host immune response. How-
ever, the line that separates bactericidal and bacteriostatic is
not as precise as previously thought. Some bacteriostatic drugs
become bactericidal when present in high concentrations at
the site of infection, or if concentrations are maintained above
the MIC for the entire dosing interval. Even macrolides and
chloramphenicol, which are traditionally considered as bac-
teriostatic drugs, can be bactericidal against some organisms.
A drug may be bactericidal against gram-positive cocci, but
bacteriostatic against gram-negative bacilli. Therefore, from a
clinical perspective, it is more useful to describe drugs as time
versus concentration-dependent, rather than as bactericidal or
bacteriostatic. This allows the clinician to consider the dosage
regimen and dosing interval to optimize therapy. There are rare
instances where a bactericidal agent is preferred over a bacte-
riostatic one. It may be preferable to prescribe a bactericidal
drug to treat life-threatening infections (such as vegetations in
endocarditis or in a systemically immunocompromised host).7
Antibacterial drugs have also been classified for veterinary
use as first-line, second-line, or third-line drugs.8 First-line
drugs are those that could be used for empirical selection in
the absence of or pending the results of culture and suscepti-
bility testing, and include amoxicillin, cephalexin, doxycycline,
and trimethoprim-sulfonamides. Second-line drugs are those to
be used on the basis of culture and susceptibility testing and
because of the lack of any appropriate first-line options. These
include ticarcillin, piperacillin, amikacin, and third-generation
cephalosporins. Fluoroquinolones were also included in this
group because in human medicine, excessive fluoroquinolone
use has been associated with emergence of antimicrobial resis-
tance and treatment failures.9 Fluoroquinolones could be con-
sidered as first-line drugs for dogs and cats suspected to have
serious gram-negative bacterial infections that require treatment
pending the results of culture and susceptibility testing. The use
of third-line drugs, including vancomycin, linezolid, and car-
bapenems such as imipenem and meropenem, is usually reserved
for situations when certain criteria are met8:
1. Infection must be documented based on clinical abnormali-
ties and culture.
2. The infection is serious and has the potential to be life-
threatening if left untreated.
3. Resistance is documented to all other reasonable first- and
second-line options.
4. The infection is potentially treatable.
5. The clinician may seek advice from an infectious disease clini-
cian or a clinical microbiologist to discuss antimicrobial sus-
ceptibility test results, and to discuss the use of these agents if
there is unfamiliarity with their use. In some instances, there
may be other viable options (e.g., topical therapy).
Pharmacodynamics of Antimicrobial Drugs
Pharmacokinetic and pharmacodynamic parameters (PK-PD
exposure relationships) are important determinants of the effi-
cacy of AMDs and serve as the basis for determination of clini-
cally effective dosage regimens and susceptibility breakpoints
and for the development of guidelines for AMD use for specific
types of infections.10
The killing of microbes by AMDs may be classified as con-
centration dependent or time dependent. AMDs that exhibit
48 SECTION 2  Antiinfective Therapy
TABLE 6-1
Classification of Antimicrobial Drugs Based on Their Mechanism of Action
Mechanism of Action Antimicrobial Drug
Inhibition of cell wall synthesis β-Lactams, glycopeptides
Protein synthesis inhibition Tetracyclines, aminoglycosides, chlor-
amphenicol, lincosamides, macrolides
Inhibition of DNA replication Fluoroquinolones
Inhibition of folic acid metabolism Trimethoprim-sulfonamides
FIGURE 6-1  Sites of action of antimicrobial drugs within the bacterial cell.
concentration-dependent killing are fluoroquinolones and
aminoglycosides. These drugs work optimally when the peak
concentration in the plasma (Cmax), or the area under the
curve (AUC) exceeds the MIC by a defined factor (Figure 6-2).
These are typically bactericidal agents and the ability to kill
bacteria increases as the AMD concentrations increase. Target
concentrations have been defined for laboratory animals and
humans and have been extrapolated to veterinary patients.
For example, the peak concentration to MIC ratio (Cmax/MIC)
should exceed 8 to 10 for optimum dosing of aminoglycosides.
The AUC for a 24-hour interval to MIC ratio (AUC24/MIC)
should exceed 100 for fluoroquinolones. For some infections,
a ratio of 125 is better, and in some patients (e.g., immuno-
compromised patients with life-threatening infections), a ratio
of 250 may be desirable. Drugs that exhibit concentration-
dependent killing also have postantibiotic effect (PAE). The
PAE is the persistence of antimicrobial effects after drug con-
centrations at the site of infection fall below the MIC. The
exact cause of the PAE varies with the drug, but it may result
Examples
Penicillin, cephalexin, meropenem, vancomycin
Doxycycline, gentamicin, erythromycin,
­clindamycin, azithromycin
Enrofloxacin, marbofloxacin
Trimethoprim-sulfamethoxazole
from irreversible binding of a drug to the target site. The
duration of the PAE is influenced by the duration of antibiotic
exposure, the drug concentration, the bacterial species pres-
ent, and the class of antibiotic. For concentration-dependent
antibiotics, administration of the total daily dose as a single
dose every 24 hours is preferred to a smaller divided dose, in
order to maximize Cmax or AUC. For aminoglycosides, this
also reduces drug toxicity.7
β-lactams (penicillins and cephalosporins), macrolides,
and lincosamides (clindamycin) exhibit time-dependent kill-
ing. As concentrations of these drugs increase, bacterial kill-
ing plateaus, and outcome is correlated with the time that the
AMD concentration spends above the MIC at the site of infec-
tion. This is usually expressed as the percent of time above the
MIC during a 24-hour interval (T > MIC). For β-lactam drugs
apart from carbapenems, PAEs are minimal. As a result, these
drugs work best when administered multiple times a day or
as continuous-rate infusions, in order to maintain drug con-
centrations at the site of infection. For some drugs (e.g., some

A serum drug concentration for poorly lipophilic agents such as
B is low. Water-soluble AMDs that are excreted in active form by
FIGURE 6-2  A, Antimicrobial drug (AMD) pharmacodynamics. Concentration-­
dependent drugs are most active when the peak concentration (Cmax), or the area-under-
the-curve (AUC) in the plasma exceeds the MIC by a defined factor. For time-dependent
drugs, outcome is correlated with the time that the AMD concentration at the site of
infection spends above the MIC. This is usually expressed as the percent of time above the
MIC during a 24-hour interval (T > MIC). B, As AMD concentrations increase, the ability
of concentration-dependent drugs such as fluoroquinolones (in this case, ciprofloxacin)
to kill bacteria increases. As concentrations of time-dependent drugs such as β-lactam
drugs (in this case, ticarcillin) increase, bacterial killing plateaus. MIC, minimum inhibitory
concentration.
cephalosporins), a long T > MIC is achieved by virtue of a long
half-life. The required time above the MIC for an AMD varies
with the pathogen present, the drug, and the site of infection,
but in general it should be 40% to 50% of the dosing inter-
val.7,10 Drug dosages that achieve concentrations in excess of
an MIC for these periods can be calculated based on pharma-
cokinetic data. For time-dependent AMD with long half-lives
(such as macrolides, clindamycin, and tetracyclines), efficacy is
determined primarily by the extent to which the area under the
curve exceeds the MIC. As the MIC of an organism increases,
it becomes more difficult to achieve these PK-PD targets, and
organisms are regarded as resistant.
CHAPTER 6  Principles of Antiinfective Therapy 49
In animals with renal failure, AMD clearance may be greatly
diminished. In this case, T > MIC and AUC/MIC are achieved
with a reduced dose or less frequent dosing. For β-lactam anti-
biotics, it is logical to increase the dose interval. For fluoroqui-
nolones, it is better to decrease the dose.
Site of Infection
In general, the concentration of an AMD at the site of infec-
tion should at least equal the MIC for the infecting organism,
but the exact concentration and the duration of the concentra-
tion at the site varies with the class of antimicrobial. For some
AMDs, the serum or plasma total (bound and unbound) drug
concentrations may not reflect the drug concentration in tissues.
Examples are the highly lipophilic macrolide antibiotics (e.g.,
azithromycin) for which the tissue drug concentration greatly
overestimates the plasma drug concentration. On the other
hand, tissue concentrations greatly underestimate the plasma or
aminoglycosides or β-lactams. Antimicrobial selection for dif-
ferent body sites is outlined in Box 6-1.
The concentration of an AMD at the site of infection is
affected by factors such as lipid solubility, protein binding, and
other factors reducing permeability, such as the presence of
large amounts of pus, scar tissue, foreign material, devitalized
tissue, and bone. Lipid-soluble drugs such as chloramphenicol,
rifampin, fluoroquinolones, and trimethoprim are most adept at
crossing membranes, including the blood-brain, alveolar, and
prostatic barriers. In contrast, poorly lipophilic, polar drugs
such as penicillins and aminoglycosides do not cross lipid mem-
branes and will not achieve adequate tissue levels in tissues in
which there is a barrier to penetration (e.g., prostate, brain, eye,
or intracellular sites). In human patients, aminoglycosides and
amphotericin B have been administered intrathecally to treat cen-
tral nervous system infections.11,12 In order to use β-lactam anti-
biotics to treat these infections, high doses are required because
of a poorly penetrated blood-brain barrier. Even in the face of
inflammation, the brain/blood concentration ratio of these drugs
the liver and concentrated in bile, such as doxycycline and ampi-
cillin, make excellent choices for susceptible hepatobiliary infec-
tions (see Chapter 88). Urinary tract infections are ideally treated
with drugs such as amoxicillin and trimethoprim-sulfonamides,
which are highly concentrated in the urine (see Chapter 89).
Other Host Factors
Other host factors that should be considered in AMD selection
and route of administration include the following:
1. History of adverse drug reactions. These are most commonly
gastrointestinal in dogs and cats, but may include hypersen-
sitivity reactions.
2. Age. With the exception of doxycycline, the use of systemic
tetracyclines is contraindicated in young animals because of
the potential for teeth discoloration (see Figure 8-5). Fluo-
roquinolones have the potential to cause cartilage and joint
toxicity in dogs between the ages of 7 and 28 weeks. The
clinical significance of this has been questioned.13,14
3. Species. Dogs and cats differ in their susceptibility to adverse
drug reactions. For example, dogs can develop severe cutane-
ous reactions to 5-flucytosine. Cats are susceptible to acute
retinal degeneration following treatment with high doses of
50 SECTION 2  Antiinfective Therapy

7. Pregnancy. Penicillins, cephalosporins, aminoglycosides,

BOX 6-1 and macrolides are generally considered safe during preg-
nancy. The volume of distribution in pregnancy is greater,
Examples of Appropriate Initial Antimicrobial Drug Choices and so higher doses may be required to achieve equivalent
for Treatment of Bacterial Infections in Different Anatomic serum concentrations.
Sites* 8. Renal and hepatic function. The dose of AMDs that are
dependent on renal elimination may require reduction in
Skin these animals with impaired renal function in order to mini-
Cephalexin mize toxicity. For example, fluoroquinolones may be more
Amoxicillin-clavulanate likely to cause seizures or retinal toxicity in animals with
Clindamycin renal failure. The use of nephrotoxic drugs such as ampho-
tericin B or aminoglycosides may be relatively contraindi-
Urinary Tract cated in animals with renal dysfunction. Adverse effects may
Amoxicillin be more common when drugs that require hepatic metabo-
Trimethoprim-sulfamethoxazole lism, such as metronidazole or chloramphenicol, are admin-
istered to animals with impaired liver function.
Prostate
Trimethoprim-sulfamethoxazole Route of Administration
Respiratory tract The most common routes of administration for AMDs in dogs and
Doxycycline cats are topical, intravenous, intramuscular, subcutaneous, and
Fluoroquinolones oral. For localized infections of the skin, eye, or ear canal, topical
administration has the advantage of delivering a high concentra-
Intestinal Tract (e.g., for animals with hepatic tion of the AMD to the site, which may overcome bacterial resis-
encephalopathy) tance mechanisms, and avoid any systemic exposure to the drug.
Ampicillin Parenteral administration, especially intravenous administration,
Neomycin provides maximum bioavailability and is recommended for (1)
treatment of life-threatening infections, (2) systemic administration
Biliary Tree of antimicrobials with poor oral bioavailability (such as amino-
Amoxicillin-clavulanate glycosides), and (3) when gastrointestinal signs or malabsorption
Doxycycline preclude effective drug administration via the oral route. Because
Fluoroquinolones parenteral AMDs are generally administered in hospital, compli-
ance with this mode of administration is also likely to be greater.
Brain Absorption may be delayed after subcutaneous or intramuscular
Metronidazole administration when perfusion of these tissues is impaired, and
Fluoroquinolones so these routes are not recommended for use when intravenous
Trimethoprim-sulfonamides administration is possible. Peak plasma drug concentrations are
achieved quickly after intravenous bolus administration. There-
Bone fore, bolus administration is an optimum approach for a concen-
Clindamycin tration-dependent antimicrobial provided rapid injection does not
Cephalosporins produce toxicity (such as may occur with fluoroquinolones). For
Amoxicillin-clavulanate a time-dependent drug such as a β-lactam, a slow infusion (even a
slow constant-rate infusion) optimizes the time-dependent activity.
*The identity of the pathogen present must also be used to guide anti- Provided gastrointestinal function is healthy, administration
microbial drug selection. of many oral AMDs results in acceptable bioavailability, even
though many popular antimicrobials have oral absorption that
is far less than 100%. Drugs with the highest oral bioavailability
enrofloxacin. Cats also are prone to esophageal ulceration in dogs and cats are the fluoroquinolones; β-lactams have low
from oral doxycycline hyclate or clindamycin unless the and variable bioavailability. Nevertheless, once the animal can
medication is administered with a bolus of water or food. tolerate oral medications and its clinical condition becomes sta-
4. Breed. Doberman pinschers may be more susceptible to hyper- ble, a switch can be made from parenteral to oral antimicrobials.
sensitivity reactions following trimethoprim-­sulfonamide Peak plasma concentrations are always lower with oral admin-
administration. istration than those achieved with intravenous administration,
5. Gastric acidity. The absorption of some AMDs, such as but for time-dependent drugs or for drugs that achieve efficacy
ketoconazole and itraconazole, is impaired by medications on the basis of an AUC/MIC exposure relationship, this route is
that suppress gastric acid production. acceptable for a cure. Early switch from intravenous to oral ther-
6. Concurrent medications. Drug interactions, such as concur- apy in human patients has gained increased favor in recent years
rent use of drugs that require metabolism using cytochrome because of reduced lengths of hospitalization, lowered costs,
P450 enzymes, can affect the choice or dose of AMD used. fewer intravascular catheter-related infections, and decreased
For example, chloramphenicol and ketoconazole are well- selective pressure on nosocomial bacterial infections.15,16 The
known P450 enzyme inhibitors, whereas rifampin is an oral bioavailability of some drugs can be maximized if they are
enzyme inducer. administered with food (e.g., clavulanic acid-amoxicillin) or
 CHAPTER 6  Principles of Antiinfective Therapy
FIGURE 6-3  Genetic mechanisms of resistance in bacteria.
without food (e.g., azithromycin). Some drugs are simply not
absorbed orally and must be administered parenterally or by
the topical route. These drugs include penicillin G, piperacillin,
ticarcillin, aminoglycosides, many cephalosporins (cefazolin,
cefotaxime, cefoxitin), meropenem, imipenem, and vancomy-
cin. Some drugs are not absorbed orally unless administered as
a prodrug. An example is cefpodoxime proxetil. The proxetil
portion of the molecule is cleaved off at the time of intestinal
absorption to allow for adequate systemic concentrations.17
Antimicrobial Resistance
Infectious agents may be intrinsically resistant to an AMD, or
be resistant as a result of genetic variations. Intrinsic or innate
resistance refers to the innate ability of an organism to resist the
effects of an AMD owing to structural or functional characteris-
tics of that organism. For example, enterococci are intrinsically
resistant to cephalosporins, because they lack the penicillin-
binding proteins that bind these antibiotics.18 Anaerobic bacte-
ria are intrinsically resistant to aminoglycosides because oxygen
is required for entry of the drug to the bacteria. The microbiol-
ogy laboratory may “suppress” the results of susceptibility tests
for antimicrobials to which organisms are intrinsically resistant
(i.e., they are not listed in the susceptibility report to the cli-
nician). Resistance can also be defined via pharmacokinetic-
pharmacodynamic targets. For example, an organism may not
express complete resistance to an antimicrobial, but the organ-
ism is effectively resistant when standard dosage regimens can-
not reach a particular peak concentration or AUC.
Genetic resistance to an AMD may occur as a result of
point mutations, DNA rearrangements, or acquisition of for-
eign DNA. Point mutations can result in alteration of the target
site of an AMD, such as the susceptibility of bacterial DNA
gyrase to fluoroquinolones.19 DNA rearrangements may include
inversions, deletions, duplications, insertions, or transpositions
(including chromosome to plasmid) of large segments of DNA.
51
The single most important mechanism of antibacterial resis-
tance is acquisition of foreign DNA by horizontal transfer, espe-
cially that carried by plasmids and transposons.20 Plasmids are
circular, double-stranded DNA molecules that are capable of
independent replication and can be transferred from one bacte-
rial strain or species to another through the process of bacterial
conjugation. Transposons are small, mobile segments of DNA
that are flanked by inverted repeats and encode one or more
resistance genes (Figure 6-3). They depend on the chromosome
or plasmids for replication. Plasmid-associated genes that con-
fer AMD resistance are frequently on transposons and can move
from the plasmid to the bacterial chromosome. A single plasmid
may contain resistance genes for more than five different AMDs.
Mechanisms of Antimicrobial Resistance
Antimicrobial Inactivation
Bacterial enzymes capable of inactivation of AMDs include
β-lactamase enzymes, enzymes that modify aminoglycoside
structure, chloramphenicol acetyltransferase, and erythromycin
esterase. β-Lactamase production is the most important mecha-
nism of resistance to β-lactam antibiotics among gram-negative
bacteria.20 A vast array of β-lactamase enzymes have been dis-
covered, which have been extensively classified into groups.21,22
Extended-spectrum β-lactamases (ESBLs) can hydrolyze not
only penicillins and first-generation cephalosporins, but also
extended-spectrum cephalosporins such as cefotaxime and cef-
tazidime (which contain an oximino group). To date, ESBLs
have only been described in gram-negative bacilli and are most
commonly produced by Escherichia coli and Klebsiella pneu-
moniae. ESBLs are often inhibited by β-lactamase inhibitors
such as clavulanic acid. The ESBL with the most importance at
the present time for small animals are the CTX-M β-lactamases,
which hydrolyze cefotaxime and other third-generation cepha-
losporins. Other types of β-lactamase enzymes include AmpC
β-lactamases, which are encoded by chromosomal genes, and
metallo-β-lactamases.20,23 These types of β-lactamase enzymes
52 SECTION 2  Antiinfective Therapy
FIGURE 6-4  Synergistic, antagonistic, and additive effects of antimicrobial drug combinations. Drugs used are referred to using letters of the alphabet. Control = no drug.
are resistant to clavulanic acid. Metallo-β-lactamases can inacti-
vate carbapenems such as meropenem and imipenem.
Membrane Alteration
Protein channels called porins facilitate the passage of hydro-
philic AMDs into gram-negative bacteria. Acquisition of porin
mutations can result in conformational changes of or reduced
expression of porins. The result is exclusion of AMDs such as
β-lactams and fluoroquinolones.24
Efflux Pump Induction
Expression of efflux pumps by bacteria allows them to exclude
certain AMDs. This is a major mechanism of resistance to tetra-
cyclines in gram-negative bacteria. A huge variety of efflux pumps
have been described.25 Efflux pumps may not be specific for one
particular class of antimicrobial. Therefore, these have been termed
“multidrug” efflux pumps and mediate multidrug resistance
(MDR). The acquisition of MDR mechanisms greatly reduces the
effective options for therapy. Multidrug-resistant organisms such
as Pseudomonas aeruginosa and Acinetobacter baumannii can
utilize multidrug efflux pumps in concert with a variety of other
resistance mechanisms, including β-lactamase enzyme production,
drug target alterations, and AMD modification enzymes.
Target Modification
Methylation of ribosomal targets by bacterial methyltransfer-
ases can lead to resistance to tetracyclines, lincosamides, ami-
noglycosides, and especially macrolide antibiotics. Expression
of altered penicillin binding proteins (PBPs) is an important
mechanism of resistance to β-lactam antimicrobials. Expres-
sion of PBP2a by staphylococci, which is encoded by the mecA
gene, confers resistance to all β-lactam antimicrobials, including
β-lactamase resistant penicillins such as methicillin and oxacillin
(methicillin-resistant staphylococci). Mutations of DNA gyrase
can confer resistance to fluoroquinolones.20 Resistance to trim-
ethoprim sulfonamides can result from altered dihydropteroate
synthetase and dihydrofolate reductase enzymes.26
Novel Approaches to Antimicrobial
Drug Resistance
With rapid spread of multidrug-resistant bacterial pathogens,
alternatives to antibiotics have been investigated. One approach
is bacteriophage therapy. Bacteriophages are viruses that infect
bacteria, and because of their specificity for certain bacterial spe-
cies, they can deliver targets to one type of organism without
harming others.27,28 Bacteriophage treatment may also be more
successful than AMD treatment in the presence of biofilms, which
impair antibiotic penetration. Other approaches that are under
investigation include use of bacterial cell wall hydrolases, cationic
antimicrobial peptides, and antisense antibiotics. Cationic anti-
microbial peptides are derived from eukaryotic cells, have immu-
nomodulatory properties, and rapidly kill a variety of bacteria.29
Although it is difficult for bacteria to develop resistance to these
peptides, multiple resistance mechanisms have been described.
Antisense antibiotics are oligonucleotides that bind to the DNA
of pathogenic microorganisms and inhibit gene expression.30
Antimicrobial Drug Combinations
When an infectious agent is suspected but has not yet been identi-
fied, the use of AMD combinations is tempting because of the added
broad spectrum of coverage that they provide. However, use of
AMD combinations does not necessarily reduce the chance of selec-
tion for a resistant organism, adds expense, and may predispose to
drug toxicity. Theoretically, drug combinations may have additive,
synergistic, or antagonistic effects ­(Figure 6-4). These properties are
not as well established in clinical situations. For example, antago-
nism between a bacteriostatic antibiotic (such as tetracycline) and
a bactericidal antibiotic that requires bacterial growth for efficacy
(such as a β-lactam) is also theoretically possible, but there is only
one clinical example of this occurrence, published in the 1950s.31
Such antagonism has not been replicated since that time.
Several reasons have been suggested for the use of AMD
combinations:
1. Some drugs must be administered in combination with other
drugs because of rapid development of resistance to them
when they are used as a sole agent. Examples are 5-flucytosine
and rifampin.
2. If a polymicrobial infection is present, one drug may not
have a sufficient spectrum of activity.
3. If the nature of an infection is not known and infection is
life-threatening, it is reasonable to commence treatment with
a broad-spectrum combination (such as a fluoroquinolone
and a β-lactam), based on knowledge of the likely pathogen
present and local prevalence of AMD resistance, pending the
results of culture and susceptibility testing. Once the results
 CHAPTER 6  Principles of Antiinfective Therapy
of culture and susceptibility testing are available, deescala-
tion should be performed.
4. Certain combinations of AMD are synergistic for treatment
of specific infections. For example, the combination of a
β-lactam and an aminoglycoside is synergistic for treatment
of enterococcal endocarditis. The relatively slow bactericidal
activity of the β-lactam is greatly enhanced by the addition
of the aminoglycoside, such that shorter durations of treat-
ment or extension of the dosing interval becomes possible
without affecting outcome.7
5. Some drugs are not effective against anaerobic bacteria (e.g.,
fluoroquinolones with the exception of pradofloxacin). They
should be combined with an agent active against anaerobes
(e.g., clindamycin or metronidazole) when a mixed infection
with anaerobic and aerobic bacteria is suspected.
6. When a vector-borne pathogen is suspected, doxycycline
may be administered until the results of further diagnos-
tic tests confirm or eliminate this suspicion. In these cases,
patients are frequently administered doxycycline in combi-
nation with another active agent to treat alternative bacterial
infections that have not yet been ruled out.
Monitoring the Response to Treatment
The response to antimicrobial treatment is best monitored based
on clinical assessment, which includes follow-up cytologic
examination or cultures. Therapeutic drug monitoring can be
used to ensure adequate serum drug concentrations when treat-
ing with aminoglycosides, vancomycin, and azole antifungal
drugs. It should be remembered that in some cases, infections
resolve because of the host immune response alone.
SUGGESTED READINGS
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J Antimicrob Chemother. 2009;64(suppl 1):i3-i10.
Levison ME, Levison JH. Pharmacokinetics and pharmacodynamics of
antibacterial agents. Infect Dis Clin North Am. 2009;23(4):791-815.
Weese JS. Investigation of antimicrobial use and the impact of antimi-
crobial use guidelines in a small animal veterinary teaching hospital:
1995-2004. J Am Vet Med Assoc. 2006;228(4):553-558.
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2. Weese JS, Blondeau JM, Boothe D, et  al. Antimicrobial use
guidelines for treatment of urinary tract disease in dogs and
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3. American Association of Feline Practitioners. Basic guidelines of
judicious therapeutic use of antimicrobials in cats. 2001. http://www.
catvets.com/uploads/PDF/antimicrobials.pdf. Last accessed May
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4. American Veterinary Medical Association. American Association
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5. Morley PS, Apley MD, Besser TE, et al. Antimicrobial drug use in
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Last accessed May 12, 2012.
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7. Levison ME, Levison JH. Pharmacokinetics and pharmacodynamics of
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10. Jacobs MR. Optimisation of antimicrobial therapy using phar-
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13. Rosanova MT, Lede R, Capurro H, et al. Assessing fluoroquinolones
as risk factor for musculoskeletal disorders in children: a systematic
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14. Sansone JM, Wilsman NJ, Leiferman EM, et al. The effect of fluo-
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21. Bush K, Jacoby GA. Updated functional classification of beta-­
lactamases. Antimicrob Agents Chemother. 2010;54:969-976.
22. Jacoby GA, Munoz-Price LS. The new beta-lactamases. N Engl
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24. Pages JM, James CE, Winterhalter M. The porin and the permeat-
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28. O’Flaherty S, Ross RP, Coffey A. Bacteriophage and their lysins
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29. Findlay B, Zhanel GG, Schweizer F. Cationic amphiphiles, a
new generation of antimicrobials inspired by the natural anti-
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31. Lepper MH, Dowling HF. Treatment of pneumococcic meningitis
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CHAPTER 7
Antiviral and Immunomodulatory Drugs
Jane E. Sykes and Mark G. Papich
w KEY POINTS
• M  ost antiviral drugs available for treatment of canine and feline
viral infections are nucleoside analogues with greatest activity
against herpesvirus and retrovirus infections.
• Because antiviral drugs also affect the function of host cell
machinery, they have considerable potential for toxicity.
• Antiviral drugs are widely used in human medicine for treat-
ment of herpesvirus, HIV and other viral infections. Much less
is known about these medications in dogs and cats and there
are few diseases of dogs and cats for which efficacy has been
demonstrated.
• Because of important differences between human and ani-
mal virus infections, one should never assume that an antiviral
agent used in people should be used in animals unless there is
proof of safety and efficacy.
• Antiviral drugs used to treat feline herpesviral infections
include famciclovir, idoxuridine, and cidofovir. Zidovudine is
INTRODUCTION
The purpose of this chapter is to describe the mechanisms of
action, spectrum of activity, and adverse effects of the major anti-
viral and immunomodulatory drugs that have exhibited utility or
have potential for treatment of companion animal infectious dis-
eases. An enormous number of different compounds have been
considered or evaluated in vitro and in vivo with variable success.
Only those currently in use for treatment of dogs and cats with
naturally occurring infections, those that show considerable prom-
ise for the future, or those evaluated in controlled clinical trials are
discussed in this chapter.
Antiviral Drugs
Antiviral agents interfere with virus-specific events in replica-
tion, including viral attachment, uncoating, assembly, and
virus-directed macromolecular synthesis. As a result, antiviral
agents typically have a restricted spectrum of activity. In con-
trast to most antibacterial drugs, antiviral drugs can also affect
the function of host cell machinery and thus antiviral drugs have
a greater risk of toxicity. In addition to acute toxicities such as
marrow suppression, many antiviral agents are immunosuppres-
sive, carcinogenic, and teratogenic. Many are given topically in
order to minimize systemic toxicity. Antiviral agents are more
likely to be effective when an intact host immune response is
present. Combinations of antiviral drugs with different mecha-
nisms of action and combinations of antiviral and immunomod-
ulatory drugs are increasingly being used to treat human viral
infections, especially HIV infection, with an associated decrease
in toxicity and reduction in selection for drug-resistant mutants.
54
the only antiretroviral drug that has demonstrated efficacy for
treatment of cats, primarily for FIV infections.
• Antiviral drugs can act synergistically with immunomodulators.
• Immunomodulators include microbial products, plant-derived
immunomodulators, naturally occurring mammalian proteins,
glucocorticoids, and synthetic compounds such as pentoxifyl-
line and opioids. The effect of many of these drugs on outcome
in dogs and cats with infectious diseases has not been fully
evaluated using large, prospective, randomized, masked, con-
trolled clinical trials.
• The parenteral use of natural or recombinant human protein
immunomodulators in dogs and cats may result in the forma-
tion of neutralizing antibodies after 1 to 2 weeks of treatment,
which can cross-react with endogenous proteins.
Although all antiviral drugs used to treat small animals were
originally developed for treatment of human viral infections, the
application of these drugs to treat viral infections of small animals
has not proven straightforward. This relates to differences in the
composition of human and companion animal viruses (which
affect their susceptibility to inactivation by antiviral drugs), or
problems relating to toxicity of human antiviral drugs in dogs
and cats.
Antiviral drugs can be classified based on their spectrum of
activity (i.e., family of viruses that are inhibited) and their mode
of action. Antiviral drugs used in small animal medicine are pri-
marily effective against herpesviruses or retroviruses, and most
are nucleoside analogues. Nucleoside analogues resemble host
nucleosides, which are nitrogenous bases with an attached sugar
molecule used as a building block for the formation of DNA
or RNA (Figure 7-1). Use of nucleoside analogues by viruses
during replication leads to the formation of abnormal nucleic
acids or termination of nucleic acid synthesis. Latent viral infec-
tions are not affected because the replication of latent virus is
suspended. However, reactivation of these infections can be
reduced in frequency or prevented. Examples of nucleoside ana-
logues and their spectrum of activity are shown in Figure 7-1
and Table 7-1. Other antiviral drugs that have been used in
clinical veterinary medicine include amino acids (l-lysine) and
neuraminidase inhibitors (oseltamivir). The value of other treat-
ments such as small inhibitory RNA molecules for viral infec-
tions of small animals is also under investigation.1
Antiherpesviral Drugs
The standard antiviral treatments for herpesviral infections
in human patients are the nucleoside analogues acyclovir and
 CHAPTER 7  Antiviral and Immunomodulatory Drugs 55

penciclovir, and their prodrugs valacyclovir and famciclo-
vir, respectively. These drugs are activated by the herpesviral
enzyme thymidine kinase (TK), which phosphorylates them to
a monophosphate form. Host cell enzymes then phosphorylate
the drugs further to triphosphate forms, which concentrate in
virus-infected cells and interfere with viral DNA replication via
inhibition of the viral DNA polymerase enzyme. Because the
DNA polymerases of different herpesviruses are inhibited to
varying degrees by acyclovir triphosphate, not all herpesviral
infections are equally susceptible. Drug resistance results from
reduced viral TK activity, altered viral TK, or altered viral DNA
polymerase. The antiherpesviral drugs cidofovir, idoxuridine,
trifluridine, and vidarabine are not dependent on viral TK for
phosphorylation and so have greater host cell toxicity. In small
animal medicine, these four drugs have been used topically to
treat ocular feline herpesviral infections.

Acyclovir and Valacyclovir

A C
B D
FIGURE 7-1  Chemical structures of deoxyguanosine (a nucleoside) and antiherpes-
viral nucleoside analogues. the grey portion is missing in the nucleoside analogues. A,
Deoxyguanosine. B, Acyclovir. C, Penciclovir. D, Ganciclovir.
Acyclovir is a synthetic analogue of the purine nucleoside deoxy-
guanosine (see Figure 7-1). In human patients, acyclovir is widely
used to treat herpes simplex and varicella-zoster virus infections,
both of which are α-herpesviruses. In small animal medicine,
acyclovir has primarily been used to treat feline herpesvirus-1
(FHV-1) infections, although FHV-1 has a much lower suscepti-
bility to the drug compared with the human herpesviruses.2 Acy-
clovir has also been used to treat canine herpesvirus-1 (CHV-1)
infections in puppies (see Chapter 16). Other nucleoside ana-
logues (trifluridine, idoxuridine, cidofovir, ganciclovir, and
penciclovir) show much greater activity than acyclovir against
FHV-1 in vitro.2-4
Topical acyclovir has been used with limited success to treat
feline herpetic dermatitis5 and keratitis.6 Topical treatment
requires frequent (>4 times a day) application for beneficial
effect.6 The oral bioavailability of acyclovir is low in cats, and
high doses are required to achieve adequate serum drug con-
centrations.7 Valacyclovir, the prodrug for acyclovir, is rapidly
converted to acyclovir by a first-pass effect after oral adminis-
tration, and administration of the prodrug results in improved
oral bioavailability of acyclovir. Unfortunately, administration
of high doses of acyclovir or valacyclovir to cats has resulted in
significant toxicity, including myelosuppression, renal tubular

TABLE 7-1
Antiviral Drugs in Use in Small Animals

Antiviral Drug Mechanism of Action Human Applications Small Animal Applications

Acyclovir/­ Guanosine analogue; interferes with Herpesviruses, especially HSV Poorly effective against FHV-1
valacyclovir viral DNA polymerase and DNA and varicella-zoster virus
synthesis. Activity requires viral TK.
Penciclovir/­ See acyclovir Herpesviruses, especially HSV FHV-1 infections
famciclovir and varicella-zoster virus
Cidofovir Deoxycytidine monophosphate Systemically to treat cytomegalo- Topical treatment of FHV-1
­analogue. Activity independent of virus ­retinitis; topically to treat ocular infections
viral TK. ­papillomavirus infections
Idoxuridine Iodinated thymidine analogue. Topical treatment of HSV kerato- Topical treatment of FHV-1
­Interferes with viral DNA ­synthesis. conjunctivitis keratitis
Trifluridine Fluorinated thymidine analogue. See idoxuridine See idoxuridine
­Interferes with viral DNA ­synthesis.
Vidarabine Adenosine analogue. Interferes with See idoxuridine See idoxuridine
viral DNA synthesis.
Zidovudine Thymidine analogue. Interferes with Systemic treatment of HIV Systemic treatment of FIV and
viral DNA synthesis. ­infections FeLV infections

FHV-1, feline herpesvirus-1; HSV, herpes simplex virus; TK, thymidine kinase.
56 SECTION 2  Antiinfective Therapy
necrosis, and hepatic necrosis, without effective suppression
of viral replication.8 Thus the use of systemic acyclovir and
valacyclovir to treat cats with herpesvirus infections is not
recommended.
Penciclovir and Famciclovir
Of all antiviral drugs used to treat small animal patients,
­penciclovir and famciclovir have shown the greatest promise.
Penciclovir is another guanosine analogue (see Figure 7-1). It
is present at much higher concentrations and for longer dura-
tion in cells than is acyclovir, which permits less frequent dos-
ing. In humans, the prodrug famciclovir is well absorbed orally
and rapidly converted to penciclovir, leading to increased oral
bioavailability of penciclovir. Most of the drug is eliminated
unchanged in the urine, and so dose reduction may be required
for animals with decreased kidney function. In human patients,
the concurrent administration of food decreases peak plasma
concentrations without affecting overall bioavailability. In
humans with herpes simplex virus and herpes zoster infections,
famciclovir is as effective as acyclovir. In contrast, penciclovir
(and thus famciclovir) have been shown to be potent inhibi-
tors of FHV-1 replication (which is not the case for ­acyclovir).
­Penciclovir-resistant mutants of FHV-1 with altered TK enzymes
have been described.9
Administration of famciclovir to cats at dosages comparable
to those used in other species results in much lower plasma con-
centrations and a longer time to development of peak plasma
concentrations of penciclovir when compared with other ani-
mal species, which suggests altered absorption or metabolism
of famciclovir in cats.10 High doses of famciclovir have been
required to achieve adequate plasma drug concentrations. Nev-
ertheless, famciclovir is well tolerated when administered orally
to cats at doses that produce clinical responses.5,10,11 Treat-
ment of cats with experimentally induced FHV-1 conjunctivitis
with famciclovir at 90 mg/kg PO q8h for 21 days resulted in
lower clinical and pathologic disease scores and decreased viral
shedding when compared with placebo-treated cats.11 Clinical
responses also occur in naturally infected cats with both acute
and chronic manifestations of disease with negligible adverse
effects.5 Because of saturation of the metabolism of famciclovir
to penciclovir, equivalent serum and tear penciclovir concentra-
tions can be achieved in cats with 40 or 90 mg/kg PO q8h of
famciclovir, so 40 mg/kg PO q8h is considered equally effica-
cious.12,13 Clinical improvement often occurs within a week of
treatment.
Ganciclovir
Ganciclovir resembles acyclovir except that it has an additional
hydroxymethyl group on its acyclic side chain (see Figure 7-1).
In human patients, ganciclovir is widely used specifically for
the treatment of human cytomegalovirus infections, which can
be life threatening in the immunocompromised. Cytomegalo-
viruses are β-herpesviruses. Systemic administration of ganci-
clovir to human patients is associated with a high prevalence
of adverse drug reactions, especially cytopenias and central
nervous system (CNS) signs, and so the use of ganciclovir is
limited to patients with life-threatening or sight-threatening
infections. A topical ophthalmic gel formulation of ganciclo-
vir (0.15%) is now available for treatment of keratitis caused
by herpes simplex virus-1. Ganciclovir is highly inhibitory to
FHV-1 replication in  vitro.4,14 Pharmacokinetic, safety, and
efficacy studies in cats have not yet been performed, but topical
ganciclovir holds promise for treatment of feline ocular herpes-
viral infections.
Cidofovir
Cidofovir is a nucleotide analogue of deoxycytidine mono-
phosphate. Because the drug already has a monophosphate
group, its metabolism to the active diphosphate form by host
cellular enzymes is not dependent on viral TK, and so it has
been used to treat acyclovir- and penciclovir-resistant infec-
tions. In human patients, cidofovir is primarily used to treat
cytomegalovirus retinitis. Cidofovir also has efficacy against
other DNA virus infections, such as poxvirus and papillomavi-
rus infections, and has been used topically as a cream to treat
viral warts in people. Because the oral bioavailability of cido-
fovir is extremely low (<5%), it is always administered intrave-
nously, intravitreally, or topically. Cidofovir has a prolonged
intracellular half-life, which enables infrequent parenteral dos-
ing regimens in human patients (once weekly, and then every
other week for maintenance therapy).
Like ganciclovir, cidofovir is highly active in  vitro against
FHV-1.4,14,15 Twice-daily administration of a 0.5% cidofo-
vir ophthalmic solution significantly decreases viral shedding
and the severity of clinical disease in cats with experimentally
induced ocular FHV-1 infection (Table 7-2).16 Local irritation
and scarring of the nasolacrimal duct has been reported with
topical administration of cidofovir to humans and rabbits with
keratoconjunctivitis.
Idoxuridine and Trifluridine
Idoxuridine and trifluridine are halogenated thymidine ana-
logues that interfere with the replication of FHV-1 in  vitro,
although they are more potent inhibitors of herpes simplex virus
replication.2,14 They have been used to treat herpesviral kerati-
tis in humans and in cats.17 These drugs are highly toxic when
given systemically, because host cell and viral DNA synthesis are
equally affected. Frequent topical application is required (five to
six times daily), and prolonged use can cause corneal irritation
or ulceration. Trifluridine has better corneal penetration than
idoxuridine and is available as a 1% ophthalmic solution. Unfor-
tunately, trifluridine is expensive, and ocular administration of
trifluridine is often extremely irritating to cats.18 In contrast,
topical idoxuridine administration is generally well tolerated.
Vidarabine
Vidarabine is an adenosine analogue that is phosphorylated by
host cellular enzymes to vidarabine triphosphate, which inter-
feres with DNA synthesis by both the virus and host cells. It
is effective against idoxuridine-resistant herpesviral strains
because its mechanism of action differs from that of idoxuri-
dine. It is reportedly well tolerated by cats when administered
five to six times daily to cats as a 3% ophthalmic ointment.18
Lysine
Lysine is an amino acid that interferes with herpesviral repli-
cation by a poorly understood mechanism. Antagonism of
arginine may somehow be involved, because a high lysine-to-
arginine ratio appears to be important for efficacy. However,
arginine itself was also shown to interfere with the replication
of herpes simplex virus in vitro.19
Lysine has shown efficacy when administered as tablets
to cats with FHV-1 conjunctivitis20 and, in another study, it
reduced shedding of reactivated virus by latently infected cats.21
 CHAPTER 7  Antiviral and Immunomodulatory Drugs 57

TABLE 7-2
Antiviral Drugs Used for Treatment of Feline Herpesvirus Infections
Drug Dose Interval Route Comments
Famciclovir 62.5 mg/cat, 125 mg/cat, q8h PO Reduce dose for cats with renal insufficiency.
or 40-90 mg/kg Compound suspensions are bitter and
poorly tolerated. Broken tablets are best
administered in a gel cap. Safe in kittens.
Cidofovir 0.5% solution q12h Topical ophthalmic Monitor for ocular irritation. Solution
prepared from commercial 7.5% intrave-
nous solution by dilution in sterile saline.
Store diluted solution up to 6 months
at 4°C, −20°C, and −80°C. Can also be
obtained from compounding pharmacies.
Idoxuridine 0.1% solution or 0.5% 5-6 times daily Topical ophthalmic Monitor for ocular irritation and ulcer-
ointment ation. Compounding may be required.
Trifluridine 1% solution 5-6 times daily Topical ophthalmic May be poorly tolerated.
Vidarabine 3% ointment 5-6 times daily Topical ophthalmic Well tolerated. Compounding may be
required.
Lysine 250 mg (kittens) q12h PO Questionable efficacy. Bolus administration
500 mg (cats) preferred to dietary supplementation.
Zidovudine 5 to 15 q12h PO, SC Monitor CBC weekly during treatment for
the first month, then monthly. Use low
end of the dose range in renal failure. Use
higher dose with caution.

When administered as tablets to cats in a shelter, there was no
reduction in upper respiratory tract disease.22 There was concern
that the stress of tablet administration may have contributed to
disease in these cats. However, in two other studies, dietary sup-
plementation with lysine was not effective for management of
upper respiratory tract disease in a shelter.23,24 In fact, cats that
received the lysine-supplemented diet had more severe disease
and more frequent viral shedding than cats that received a non-
supplemented ration, despite having increased plasma lysine
concentrations. The lysine-supplemented diet did not affect
plasma arginine concentration, so altered arginine levels did not
appear to contribute to the increased severity of disease.
Antiretroviral Drugs
All antiviral drugs used to treat cats with retrovirus infections
have been nucleoside analogues, which inhibit the DNA poly-
merase function of the retroviral reverse transcriptase (RT)
enzyme. Unfortunately, many drugs used for treatment of HIV
infections (such as protease inhibitors) are not effective for
treatment of feline retrovirus infections, because they only act
on HIV enzymes. The only antiretroviral that has shown benefit
in naturally infected cats is zidovudine (AZT). At the time of
writing, an integrase inhibitor known as raltegravir has shown
early promise for treatment of FeLV infections both in vitro and
in vivo.25,26 Many drugs that have activity against feline retrovi-
ruses in vitro, such as ribavirin and adefovir (PMEA), are toxic
when given to cats, which limits their use in practice.
Zidovudine and Fozivudine
Zidovudine (azidothymidine; AZT; Retrovir) is a thymidine ana-
logue and was one of the first drugs shown to be effective against
HIV. Inside host cells, it is converted to the active triphosphate
form. AZT inhibits the replication of FIV, reduces plasma viral
load, improves stomatitis, and increases CD4/CD8 ratios in cats
naturally infected with FIV.27,28 Some FIV isolates are resistant as
a result of mutations in the RT enzyme. Although AZT is active
against FeLV in vitro, when compared with FIV-infected cats, it
has not performed as well for treatment of naturally infected, sick
cats with chronic FeLV infection. Nevertheless, improvement in
stomatitis, reduced antigenemia, and reduction in development of
lymphoma have been reported in studies of naturally and experi-
mentally FeLV-infected cats that were treated with AZT.28,29
AZT is available as a10 mg/mL syrup and a 10 mg/mL injec-
tion. It has good oral bioavailability and is well distributed to
tissues, including the CNS. It is metabolized to an inactive form
by the liver and excreted by the kidneys. Dosage reduction has
been recommended for cats with renal failure. Unfortunately,
some cats treated with AZT can develop dose-related hemato-
logic adverse effects, most commonly nonregenerative anemia
and neutropenia, so the CBC must be monitored during treat-
ment. Adverse effects may be confused with retrovirus-induced
cytopenias. In human patients with HIV infection, cytopenias
are more likely to occur when disease is advanced.
Fozivudine is a thioether lipid-zidovudine conjugate that
undergoes intracellular cleavage to zidovudine monophos-
phate and subsequent phosphorylation to the active triphos-
phate form. Cleavage preferentially occurs in lymphocytes and
monocytes compared with RBC and marrow stem cells, and so
hematologic toxicity is less likely to occur. Fozivudine reduces
viremia in cats experimentally infected with FIV, without signif-
icant adverse effects.30 Further study is required to evaluate this
drug for treatment of chronic FIV and FeLV infections in cats.
58 SECTION 2  Antiinfective Therapy
Lamivudine
Lamivudine (3TC), a cytidine analogue, is synergistic when
combined with AZT for treatment of HIV infection, and the
combination is in common use in human medicine. AZT/3TC
prevented FIV infection when given to cats shortly after experi-
mental inoculation, but did not appear to be beneficial for treat-
ment of cats with chronic FIV infection.31 In addition, severe
hematologic adverse effects and fever occurred in some cats.
Other Antiretroviral Drugs
Raltegravir inhibits the retroviral integrase enzyme. Integrase
incorporates transcribed viral DNA into the host chromosome.
In human patients, raltegravir is approved for treatment of HIV
infections and is used in combination with other antiretroviral
drugs. Although expensive, raltegravir has shown great promise
for treatment of FeLV infections in vitro and also in vivo.25,26
Other drugs that have shown promise in vitro for treatment of
FeLV infections, with no evidence of toxicity to cell cultures,
are the nucleoside analogues tenofovir, decitabine, and gem-
citabine.26 Tenofovir is used as part of combination therapy to
treat HIV infections. It is administered as a prodrug. Decitabine
and gemcitabine are cytidine analogues used in human patients
to treat myelodysplastic syndromes and carcinomas, respec-
tively. The use of combinations of gemcitabine and carboplatin
to treat carcinomas in cats has been reported, but cytopenias
and gastrointestinal toxicity occurred in some of the cats.32
Plerixafor is a bicyclam derivative that selectively blocks the
chemokine receptor, CXCR4. This receptor is used by FIV to
enter cells (see Chapter 21). In a placebo-controlled, masked
clinical trial, administration of plerixafor to cats with FIV
infection for 6 weeks reduced proviral load but did not lead to
improvement in clinical or immunologic variables.33
Antiinfluenza Viral Drugs
The main drugs used in human medicine to treat influenza virus
infections are neuraminidase inhibitors, such as oseltamivir and
zanamivir, and the tricyclic amines amantadine and rimanta-
dine, which inhibit the M2 ion channel protein that is present in
influenza A viruses (Figure 7-2). Only oseltamivir has been used
to any great extent in small animals. Amantadine, which also
has antagonistic effects at the NMDA (N-methyl-d-aspartate)
receptor, has been used to treat osteoarthritis in animals in com-
bination with other drugs.34
Oseltamivir
Oseltamivir (Tamiflu) is the prodrug of oseltamivir carboxylate
(GS4071), a potent inhibitor of influenza virus neuraminidase.
Oseltamivir was developed because of the poor oral bioavailabil-
ity of zanamivir, a neuraminidase inhibitor that is structurally
similar to GS4071. Neuraminidase is a surface glycoprotein of
both influenza A and influenza B viruses (see Figure 7-2). The
viral neuraminidase cleaves sialic acid residues on the surface of
infected cells, which allows new virus particles to be released from
host cells. It also prevents aggregation of virus particles after they
are released and facilitates spread of the virus through the mucus
of the respiratory tract by cleaving sialic acid residues in mucin.
In humans and dogs, oseltamivir has high oral bioavailabil-
ity.35 Esterase enzymes in the liver then convert oseltamivir to
its active form, which is well distributed to most body fluids,
including surface epithelial cells throughout the upper and
lower respiratory tract. Elimination of the drug relies on renal
FIGURE 7-2  Structure of an influenza virus. The M2 protein, which is only ­present in
influenza A viruses, is inhibited by tricyclic amines such as amantadine. ­Oseltamivir inhib-
its the viral neuraminidase.
excretion. The drug is well tolerated in humans, with gastroin-
testinal signs being the most frequently reported adverse effects.
The prevalence of resistance to oseltamivir among influ-
enza virus isolates is generally low (<5%). High-level resistance
results from mutations in the viral neuraminidase. Neuramini-
dase inhibitors could play a critical role in prevention of mor-
tality in human influenza virus pandemics, so strategies that
minimize the selection of resistant mutants are important. The
Centers for Disease Control and Prevention recommends priori-
tizing antiviral treatment to human patients at risk of complica-
tions of influenza, such as the very young and very old.36
Oseltamivir has been used to treat canine parvovirus (CPV)
enteritis in puppies, with anecdotal reports of improved ­outcome.
A single prospective, randomized, masked, ­placebo-controlled
trial of 35 dogs with CPV enteritis showed that dogs treated with
oseltamivir (2 mg/kg PO q12h) had no significant drop in their
white blood cell count, whereas untreated dogs had a significant
drop in their white blood cell count in the first 5 days of hospi-
talization.37 Treated dogs also gained weight during hospitaliza-
tion, whereas untreated dogs lost weight. However, there was
no difference in hospitalization time, treatments needed, clinical
scores, morbidity, or mortality between the two groups, and the
number of dogs in each group was small. No significant adverse
drug effects were observed, but oseltamivir was ­administered
as a 1:1 dilution with water, in order to reduce reactions to the
taste of the drug and vomiting shortly after drug administration.
The authors acknowledged that there were potential concerns
that related to administration of an oral medication to dogs
with enteritis, with variability in drug absorption.
As CPV has no neuraminidase, it was hypothesized that osel-
tamivir instead may act on the neuraminidases of bacteria that
are normally responsible for secondary bacterial infections in
CPV enteritis, which are primarily those of the gastrointesti-
nal tract. The role of bacterial neuraminidases in the pathogen-
esis of enteric bacterial infections and bacterial translocation is
unknown. Bacterial neuraminidase enzymes may play a role in
biofilm formation and help bacteria to invade mucin layers of
the respiratory tract.38 It has been suggested that gut bacteria
 CHAPTER 7  Antiviral and Immunomodulatory Drugs
may use neuraminidase enzymes to cleave sialic acid residues on
gastrointestinal epithelial cells, which exposes receptor sites for
bacterial adherence.
A major concern that relates to treatment of CPV enteri-
tis with oseltamivir is the possibility of selection for resistant
mutants among influenza viruses if widespread use of the drug
occurs in veterinary clinics. Given the restrictions on the use of
this drug for treatment of human influenza virus infections, fur-
ther investigation is required before the use of oseltamivir can
be recommended for treatment of CPV enteritis.
Other Antiviral Drugs
Although not useful for treatment of cats because of toxicity,
the nucleoside analogue ribavirin has shown promise in  vitro
for treatment of canine distemper virus (CDV) infections.
Another drug, 5-ethynyl-1-beta-d-ribofuranosylimidazole-4-
carboxamide (EICAR), has also shown activity against CDV
in  vitro39 and for treatment of measles virus, which is closely
related to CDV.40 Ribavirin is primarily used to treat hepatitis C
virus infections in humans. The pharmacokinetics and toxicity
of ribavirin and EICAR in dogs require further study.
Nelfinavir is a protease inhibitor used to treat HIV infec-
tion. It also limits the replication of severe acute respiratory
syndrome (SARS) coronavirus in vitro,41 but its mode of action
against coronaviruses is poorly understood. Nelfinavir was
not effective in a mouse model of SARS.42 Although nelfinavir
alone was not completely effective, a combination of nelfinavir
and Galanthus nivalis agglutinin (GNA) inhibited the replica-
tion of feline coronavirus in  vitro and may have application
for the treatment of feline infectious peritonitis (FIP).43 GNA
appears to bind the viral outer envelope glycoproteins and
prevent cellular entry. The use of nelfinavir was reported in a
few cats with naturally occurring coronavirus infections,43,44
but the toxicity and pharmacokinetics of nelfinavir and GMA
in cats and efficacy of these drugs for treatment of FIP remains
unknown.
Immunomodulators
An immunomodulator is any drug that alters immune system
function. Immunomodulators are also referred to as biologic
response modifiers. Their use for treatment of infectious dis-
eases may be beneficial when compromise of the immune system
impairs effective antimicrobial drug treatment. Alternatively,
immunomodulators can be used to dampen an excessive host
inflammatory response.
A variety of immunomodulators have been used to treat
feline retroviral infections, FIP, FHV-1 infections, and canine
and feline parvoviral enteritis (Table 7-3). Immunomodulators
used in veterinary medicine can be divided into six main groups:
(1) microbial products; (2) plant-derived biologic response
modifiers; (3) naturally occurring cytokines, such as colony-
stimulating factors, and interferons; (4) immunoglobulins;
(5) glucocorticoids; and (6) synthetic compounds with immuno-
modulatory activity, such as pentoxifylline.
Treatment of canine and feline viral infections with immuno-
modulators has frequently been associated with disappointing
results, and data from large, prospective, controlled clinical tri-
als are generally not available. Frequently, an immunomodula-
tor shows antiviral activity in vitro but this does not correlate
with treatment responses in  vivo. Sometimes, mild to moder-
ate clinical improvement is observed, but treatment does not
59
result in cure. The use of human recombinant cytokines beyond
1 or 2 weeks of treatment has been associated with the develop-
ment of antibodies against the cytokines, which have the poten-
tial to cross-react with endogenous cytokines. In the absence
of data from randomized, placebo-controlled clinical trials to
support their use, immunomodulators should be used with cau-
tion, because they may have the potential to accelerate disease.
This may occur for viruses that are dependent on rapidly divid-
ing cells for replication (such as parvovirus) or those that rep-
licate only in activated lymphocytes (such as FIV). Promotion
of an inflammatory response may also have detrimental effects
if immune-mediated processes play an important role in the
pathogenesis of disease (such as for FIP).
Microbial Products
Microbial products with immunomodulatory activity include
bacillus Calmette-Guérin extract (a mycobacterial cell wall
extract from Mycobacterium bovis), purified staphylococcal
protein A, killed Propionibacterium acnes (ImmunoRegulin,
ImmunoVet), Staphylococcus aureus phage lysate (Staph-
age Lysate, Delmont Laboratories, USA), Serratia marcescens
abstract, and inactivated poxviruses (Baypamun, Bayer). To
date, controlled clinical trials with these products have not dem-
onstrated significant efficacy for treatment of viral infections
in dogs and cats. Staphylococcal phage lysate, which contains
components of S. aureus and a bacteriophage, and Propionibac-
terium acnes immunotherapy have shown efficacy for treatment
of pyoderma in dogs and are available commercially for this
purpose (see Table 7-3; also see Chapter 84).45
Plant-Derived Biologic Response Modifiers
Acemannan (Carrisyn, Carrington Laboratories, Irving, TX) is
a mucopolysaccharide derived from aloe vera plant leaves that
induces cytokine production and dendritic cell maturation. It
stimulates macrophages to secrete a variety of cytokines, includ-
ing IFN-γ, TNF-α, prostaglandin E2, Il-1, and Il-6. It has been
used to treat FIV and FeLV infections, either by oral or paren-
teral administration,46,47 and a veterinary product is available
for treatment of canine and feline fibrosarcomas. Controlled
trials are needed to understand the efficacy of this treatment in
naturally infected cats.
Polyprenyl immunostimulant (Sass & Sass, Inc., Oakridge,
TN) is a plant-derived investigational veterinary biological that
is made up of phosphorylated, linear polyisoprenols. In vitro, it
upregulates synthesis of Th1 cytokines and has antiviral proper-
ties. Cats with experimentally induced FHV-1 infection had a
decreased severity and duration of upper respiratory tract signs
when treated with polyprenyl immunostimulant, in compari-
son to placebo-treated cats.47a Long-term (>2 years) survival
has been reported in a few cats with noneffusive FIP that were
treated with polyprenyl immunostimulant. The response in cats
with effusive FIP has been disappointing.48
Naturally Occurring Mammalian Proteins
Colony-Stimulating Factors
Colony-stimulating factors (CSFs) are naturally occurring gly-
coproteins that stimulate production, differentiation, survival,
and activation of white blood cells. Examples include erythro-
poietin; thrombopoietin; Il-5 (which stimulates eosinophil and
basophil production); Il-3; stem cell factor; and macrophage
CSF, granulocyte CSF (G-CSF), and granulocyte-macrophage
CSF (GM-CSF). Recombinant G-CSF has been used most
60 SECTION 2  Antiinfective Therapy

TABLE 7-3
Commercially Available Immunomodulators That May Have Benefit for Treatment of Infectious Diseases of Dogs and Cats
Drug Dose Interval Species Route Comments
Acemannan 2 mg/kg Weekly C SC For treatment of fibrosarcomas,
efficacy unclear for FIV and
FeLV infections
Feline recombinant 1 million U/kg q24h for 5 consecutive C, D SC For FeLV infections
interferon omega days on days 0, 14,
and 60
0.1 million U/kg q24 C Oromucosal For refractory caudal stomatitis
associated with FCV infection
Filgrastim 5 µg/kg q24h C, D SC Monitor CBC weekly during
treatment. Long-term use
(>2 weeks) can lead to
development of neutralizing
antibodies.
Lactoferrin 200 mg powder q24h C Topical For refractory gingivitis and
or 40 mg/kg stomatitis. Must be purchased
solution from chemical suppliers
Lymphocyte T-cell 1 mL Weekly C SC For treatment of FeLV and FIV
­immunomodulator infections
Human recombinant 104 to 106 U/kg q24h C SC Monitor CBC weekly during
interferon alpha 1 to 50 U/cat q24h PO parenteral treatment. Long-
term parenteral use can lead
to development of neutralizing
antibodies
Pentoxifylline 15 mg/kg q8h D PO Benefits for treatment of FIP
¼ of a 400 mg q8h C unclear. Co-administration
tablet of cytochrome P450 inhibi-
tors may increase serum drug
concentrations
Polyprenyl 3 mg/kg Three times weekly C PO Possible benefit for treatment of
­immunostimulant noneffusive FIP
Prednisolone 1 to 2 mg/kg q24h C PO For FIP
Propionibacterium 0.1 mg dogs <7 kg Twice weekly. After D IV For pyoderma. Available as a
acnes, killed 0.2 mg 7-20 kg 14 d, weekly ­until 0.4 mg/mL solution
­remission, and
0.4 mg 20-34 kg
monthly thereafter to
0.8 mg > 34 kg maintain remission
Staphylococcus 0.5 mL Once or twice weekly D SC For pyoderma
­aureus phage lysate

C, Cats; D, dogs.

widely in veterinary medicine as an immunomodulator. The use
of these drugs may be limited by their cost and immunogenicity.
Granulocyte colony-stimulating factor.  Granulocyte CSF
stimulates growth and differentiation of the neutrophil cell line.
It also stimulates neutrophil function, including chemotaxis,
phagocytosis, antibody-dependent cellular cytotoxicity, and
superoxide production. Nonglycosylated, recombinant human
G-CSF is available under the trade name Neupogen. Pegfil-
grastim (Neulasta) is a pegylated form with a long half-life and
single-dose administration. Lenograstim is a glycosylated form
available outside the United States. These three forms have simi-
lar efficacy and adverse effects in humans. G-CSF is approved
for use in humans for treatment of chemotherapy-induced
myelosuppression, for treatment of severe chronic neutropenia,
for mobilization and collection of peripheral blood stem cells
for bone marrow transplantation, and to accelerate myeloid
reconstitution after hematopoietic stem cell transplantation.49
G-CSF and GM-CSF have been used to treat pneumonia and
severe sepsis in humans without neutropenia, but significant
benefit has not been identified.50,51
When G-CSF is administered as a daily subcutaneous injec-
tion to dogs and cats, the neutrophil count increases over sev-
eral days. This results from an enhanced rate of granulopoiesis
and a shortened neutrophil maturation time. Slight increases
in monocyte and lymphocyte counts can also occur.52 Counts
return to normal 5 days after discontinuation of treatment.
 CHAPTER 7  Antiviral and Immunomodulatory Drugs
In veterinary medicine, filgrastim has been used to treat che-
motherapy-induced neutropenia and cyclic neutropenia in gray
collies. Filgrastim was not effective for treatment of leukopenia
in experimentally induced CPV infection.53,54 Repeated admin-
istration of filgrastim to dogs and cats has been associated with
development of antibodies to the protein within 2 to 3 weeks of
treatment, which can cross-react with endogenous G-CSF. Thus,
although it can increase neutrophil counts in some FIV-infected
cats, long-term use of filgrastim is not recommended.55 Recom-
binant canine G-CSF (rcG-CSF) may be safer for long-term use
in dogs, and possibly also cats. One recombinant canine formu-
lation mobilized neutrophils in dogs within 24 hours of admin-
istration.56 Recombinant canine G-CSF reduces the severity and
duration of bone marrow suppression induced by cyclophos-
phamide in dogs.57 In a prospective, non-randomized clinical
trial for treatment of CPV infection, neutrophil counts were
higher and hospital times were shorter in dogs treated with rcG-
CSF, but survival times were decreased.58 Treatment of canine
and feline parvovirus infections with G-CSF might cause harm,
because the increased cell turnover induced by the drug might
promote parvovirus replication. Further studies are required to
evaluate the utility of rcG-CSF for treatment of neutropenias
associated with canine parvoviral enteritis, feline retrovirus
infections, and chronic canine monocytic ehrlichiosis.
In dogs and cats, exogenous G-CSF is well tolerated. In
human patients, the main adverse effects of treatment are bone
and musculoskeletal pain, headache, anemia, thrombocytope-
nia, and splenomegaly. Long-term use in humans can be associ-
ated with osteopenia as a result of upregulation of osteoclast
activity. Rare complications in people include splenic rupture
and exacerbation of autoimmune disorders.
Granulocyte-macrophage colony-stimulating factor.  GM-CSF
stimulates growth and function of neutrophils, monocytes, and
eosinophils. Commercial GM-CSF formulations available for
treatment of hematologic disorders in human patients include
sargramostim, molgramostim, and regramostim, which vary
slightly in amino acid sequence and degree of glycosylation.
When used to treat cats with FIV, viral load increased,59 and
so the use of GM-CSF is not recommended for treatment of
FIV-induced cytopenias. Interestingly, sargramostim has been
used to treat some human patients with AIDS-associated oppor-
tunistic infections, which include oropharyngeal candidiasis
and disseminated mycobacterial infections. Administration of
recombinant human GM-CSF to dogs was associated with anti-
body development after 10 days.60
Interferons
Interferons are cytokines with antiviral properties. IFN-α, IFN-
β, and IFN-ω are type I interferons, which are produced by leu-
kocytes and fibroblasts in response to a viral infection. Type I
interferons activate natural killer (NK) cells, increase expression
of major histocompatibility complex (MHC) class I molecules,
and have antitumor activity. IFN-γ is the only type II interferon
and is produced by T lymphocytes and NK cells in response to
antigenic stimulation. IFN-γ plays a critical role in the clear-
ance of intracellular pathogens by macrophages. Recombinant
human IFN-γ has been used to treat chronic granulomatous
disease in humans as well as chronic infections caused by intra-
cellular bacteria and fungi, but recombinant veterinary prepara-
tions are not available.
Interferon alpha.  Naturally occurring IFN-α represents a
group of more than 20 molecules that vary slightly in composi-
tion and inhibit viral nucleic acid and protein synthesis. A variety
61
of recombinant human and natural IFN-α preparations are avail-
able, including pegylated forms that have prolonged biologic
half-lives. In human patients, IFN-α is used in conjunction with
antiviral drugs to treat hepatitis B and hepatitis C virus infec-
tions, papillomavirus infections, and HIV-associated Kaposi’s
sarcoma. It is generally administered for 24 or 48 weeks.
Recombinant human IFN-α (rhIFN-α) has been administered
parenterally and orally to cats for treatment of feline retroviral
infections, feline upper respiratory viral infections, and FIP. It
inhibits replication of FeLV, FIV, FHV-1, and feline coronavirus
in vitro. Unfortunately, mixed results have been obtained after par-
enteral and oral administration to cats with retrovirus infections,
and parenteral IFN-α was not effective for treatment of FIP.61 Par-
enteral administration can lead to the development of neutralizing
antibodies and apparent loss of activity after 3 or 7 weeks.62
Oral administration of a low dose of rhIFN-α to treat viral
infections of animals has been controversial because of proteo-
lytic degradation of rhIFN-α by gastric acid. Nevertheless, ben-
eficial outcomes have been reported in some cats with FHV-1,
FeLV, and FIV infections, presumably because immunomodula-
tion follows mucosal absorption of the drug. Topical application
of rhIFN-α to the eye has been used to treat FHV-1 keratitis, but
controlled studies using this treatment are lacking. With the avail-
ability of more effective antiviral drugs such as famciclovir, it is
likely that the use of these antiviral drugs may take precedence
over, or be used in combination with, treatment with IFN-α.
Feline recombinant interferon-omega.  IFN-ω is a Type 1 inter-
feron closely related to IFN-α. It is secreted by virus-infected
leukocytes. Its precise mechanism of action is not known. In
dogs it increases macrophage and NK cell activity, and it has
antiviral activity against several feline viruses in vitro, including
FeLV, FHV-1, feline calicivirus (FCV), canine and feline par-
voviruses, and feline coronavirus. It also has antitumor activ-
ity.63 A recombinant feline formulation of IFN-ω produced in
silkworms is available in Europe and Canada for veterinary use
(rfIFN-ω, Virbagen omega, Virbac). Because it has similar activ-
ity in canine and feline cells, it has potential for treatment of
both canine and feline viral infections. The optimal dosing regi-
men is unknown, but the manufacturer recommends three cycles
of treatment, on days 0, 14, and 60, for 5 consecutive days.
Preliminary data suggested that treatment of cats with
FIP with rfIFN-ω might increase survival times,64 but a
­randomized, placebo-controlled study of naturally infected
cats demonstrated no improvement in quality of life or sur-
vival time.65 When cats with naturally occurring FeLV-related
disease were treated with rfIFN-ω in a placebo-controlled trial,
survival time over a 2-month follow-up period increased.66
Improvement in clinical scores and laboratory parameters
was also documented in some sick cats that were naturally
infected with FeLV and FIV, despite no significant changes in
viral load.67 The drug also has been used topically, orally, and
parenterally to treat FHV-1 infections, and subcutaneously to
treat refractory FCV-associated stomatitis.68-71 Oromucosal
administration of rfIFN-ω to cats with refractory FCV-asso-
ciated stomatitis led to significant clinical improvement when
compared with placebo.68 Treatment of puppies with natu-
rally and experimentally induced CPV enteritis with rfIFN-ω
in a number of placebo-controlled trials was associated with
reduced disease severity and, in some studies, significantly
reduced mortality. In contrast, there was no improvement in
survival in cats from a cattery with feline panleukopenia virus
infection that were treated with rfIFN-ω when compared with
an untreated control group.72
62 SECTION 2  Antiinfective Therapy

Recombinant fIFN-ω is well tolerated by dogs and cats.

Transient lethargy, fever, vomiting, mild diarrhea, and anorexia
have been documented in some treated cats, especially at higher
doses (2.5 × 106 U/kg). Mild neutropenia, eosinophilia, and
reversible increases in the activity of serum AST have also been
described following treatment.73 Additional studies of the effi-
cacy and safety of rfIFN-ω in larger numbers of sick cats with
retroviral infections and cats and dogs with other viral infec-
tions are required.

Lymphocyte T-cell Immunomodulator

Lymphocyte T-cell immunomodulator (T-cyte Therapeu-
tics, Inc.) is a protein derived from thymus epithelial cells.
According to the product brochure, LTCI upregulates Il-2
production in vitro and regulates CD4 T-cell function. It was
also reported to stimulate platelet production in mice with
­chemotherapy-induced thrombocytopenia. LTCI has been
conditionally approved by the U.S. Department of Agriculture
as an aid for treatment of FIV and FeLV infections, and for the
associated signs of cytopenias and opportunistic infections.
Controlled independent studies of the efficacy of LTCI in cats
are required.

Immunoglobulins
In human patients, human intravenous immune globulin (IVIG)
has been used for treatment of immunodeficiencies, as well as
autoimmune disorders (Figure 7-3). A single infusion of human
IVIG has been used successfully to treat immune-mediated
thrombocytopenia in dogs,74 but repeated administration to
animals may lead to transfusion reactions. Intravenous immu-
noglobulin preparations have been commercially available for
passive immunization of cats or dogs in Europe. These contain
antibodies to FPV, FHV-1, and FCV (Feliserin, IDT Biologika
GmbH, Rodelben, Germany), or CDV, canine adenovirus, and
CPV (Stagloban, IDT Biologika GmbH). The efficacy of these
preparations has not been reported. Subcutaneous and intraper-
itoneal administration of adult cat serum to kittens with failure
of passive transfer can provide IgG concentrations equivalent to
those in kittens that suckle normally.75 In general, when given
parenterally in high doses, specific immunoglobulins are only
effective for prevention, and not treatment of an infectious dis-
ease.76 Oral administration is not effective. Passive administra-
tion of antitoxins is also used to treat tetanus (see Chapter 54)
and for neutralization of snake venom components. Passive
immunization may be associated with allergic reactions, which
can be severe, and it has the potential to interfere with subse-
quent active immunization attempts. Administered antibodies
are generally eliminated over a period of 2 to 3 weeks.
Lactoferrin
Bovine lactoferrin is a glycoprotein with immunomodulatory
properties that is normally present in exocrine secretions and
neutrophils. It reduces cytokine secretion by mononuclear cells
and lymphocyte activation in cats infected with FIV. In  vitro,
bovine lactoferrin has antiviral activity against FCV and
­FHV-1.77,78 Topical application (40 mg/kg q24h for 14 days) to
the mouth of a small number of FIV-positive and FIV-negative
cats with stomatitis was associated with improved appetite and
reduced oral inflammation, salivation, and pain, and increased
neutrophil phagocytic activity.79 Chronic oral administration of
bovine lactoferrin to a dog with β2-integrin-related neutrophil
dysfunction was associated with increased neutrophil function
and recovery from opportunistic infections.80
FIGURE 7-3  Human intravenous immune globulin solution (inverted for
administration).
Glucocorticoids
In animals and humans with infectious diseases, treatment with
glucocorticoids is sometimes used to dampen an overzealous
immune response that contributes to the pathology of infection.
Glucocorticoids have potent antiinflammatory and immunosup-
pressive properties. They suppress cytokine synthesis, decrease
antibody production, cause lymphocytolysis and eosinopenia,
decrease neutrophil margination, interfere with cyclooxygen-
ase-2, and reduce the activity of phospholipase A2, and thus
initiation of the arachidonic acid cascade. However, glucocor-
ticoid administration also has the potential to worsen outcome
through excessive immune suppression, so their use has been
advocated only for specific infectious diseases under certain
circumstances. Glucocorticoids can also contribute to develop-
ment of secondary infections even when they are used to treat
another disease. For example, it is not unusual in dogs or cats
for a latent infection to emerge, or for a secondary infection to
develop (e.g., demodicosis, staphylococcal pyoderma, dermato-
phytosis, systemic mycoses) when glucocorticoids are used to
produce immunosuppression.
An example of the beneficial effect of glucocorticoids for
treatment of an infectious disease is in FIP, where inflammation
plays a major role in pathogenesis. Although controlled clinical
trials comparing prednisone treatment with a placebo have not
been performed, for many cats, treatment with prednisolone
(1 to 2 mg/kg PO q24h) is associated with transient complete
or partial remission and as a result is generally accepted as the
standard of care for treatment of FIP (see Chapter 20).65
A short course of prednisolone using immunosuppres-
sive doses (1 mg/kg PO q12h) has also been used to suppress
secondary immune-mediated hemolysis in cats infected with
Mycoplasma haemofelis,81 but given that glucocorticoids
can reactivate hemoplasma infections, additional studies are
needed to determine whether this practice improves outcome.
Similarly, glucocorticoid treatment has been used to suppress
 CHAPTER 7  Antiviral and Immunomodulatory Drugs
immune-mediated consequences of infection with the tick-borne
pathogens Ehrlichia canis and Babesia spp, when antimicrobial
treatment alone is not followed by complete resolution of clini-
cal abnormalities. Adjunctive glucocorticoid treatment did not
worsen outcome in dogs with Rocky Mountain spotted fever.82
In contrast, glucocorticoid administration may have a negative
impact in dogs with Lyme arthritis.83 There are anecdotal reports
of improved outcome for dogs with suspected Lyme glomerulo-
nephritis that are treated with methylprednisolone sodium suc-
cinate in conjunction with other immunosuppressive drugs, such
as mycophenolate mofetil or azathioprine (see Chapter 51).
Many large human studies have assessed the role of glucocor-
ticoid treatment in septic shock (see Chapter 86). Treatment with
high doses of hydrocortisone for less than 5 days is contraindi-
cated because of secondary infection and gastrointestinal bleed-
ing. Treatment with physiologic doses of glucocorticoids has been
advocated to overcome relative adrenal insufficiency, which occurs
in severe septic shock in both humans and dogs.84 Recent consen-
sus guidelines for human patients with septic shock recommend
adjunctive hydrocortisone treatment in patients who are unre-
sponsive to fluid resuscitation and treatment with vasopressors.85
Infections that generate inflammation within the eye and
CNS are frequently treated with anti-inflammatory doses of
topical or systemic glucocorticoids, respectively, in conjunc-
tion with antimicrobial drugs. The goal of such treatment is to
minimize or prevent loss of vision or deterioration of neurologic
status, especially when organism death is followed by a pro-
found inflammatory response. For CNS infections, the effect of
concurrent glucocorticoid treatment may depend on the specific
pathogen present and the immune response of the infected indi-
vidual. Prospective studies that evaluate outcome after adjunc-
tive glucocorticoid treatment in CNS infections of dogs and cats
are lacking. In a retrospective study, treatment of dogs and cats
with CNS cryptococcosis with antiinflammatory doses of glu-
cocorticoids increased survival in the first 15 days after starting
antifungal drug treatment.86
Other
Pentoxifylline
Pentoxifylline is a synthetic methylxanthine derived from theo-
bromine. It is a broad-spectrum phosphodiesterase inhibitor
with a variety of immunomodulatory properties. In rodents and
humans it inhibits TNF-α, Il-1, Il-6, and Il-12 production, as
well as the activation of T and B cells, and increases neutrophil
mobility and chemotaxis. It increases erythrocyte cAMP levels,
which results in increased erythrocyte deformability, lowered
blood viscosity, and improved microcirculation and tissue per-
fusion. Pentoxifylline also decreases platelet aggregation, and
increases tissue plasminogen activator in patients with hyperag-
gregable platelets, so may be useful for treatment of disorders
that result in hypercoagulable states. The value of pentoxifyl-
line for treatment of endotoxemia has been primarily studied
in horses. Despite significant decreases in TNF and IL-6 activ-
ity after incubation of equine monocytes with pentoxifylline
in vitro, these effects did not occur in horses in vivo.87 Beneficial
effects of pentoxifylline are limited in horses when administered
IV after challenge with endotoxin.88
In dogs, pentoxifylline has been used to treat a variety of
skin diseases, including canine atopic dermatitis, dermatomy-
ositis, and cutaneous vasculopathies.89,90 In cats it has been
used to treat FIP, but a masked, placebo-controlled study of the
related drug propentofylline showed no benefit of treatment.91
63
In dogs, pentoxifylline has a low and unpredictable oral bio-
availability and a short half-life, and so three-times-daily
administration has been recommended. Adverse effects include
vomiting and decreased appetite, which are more likely to
occur when the tablets are crushed. Crushed tablets are also
unpalatable to cats.
Antimicrobial Drugs with Immunomodulatory Properties
Antimicrobial drugs with immunomodulatory properties
include the macrolides, doxycycline, and metronidazole. The
mechanisms of these activities are discussed in Chapter 8.
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47a. Legendre A. Personal communication. 2011.
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52. Fulton R, Gasper PW, Ogilvie GK, et al. Effect of recombi-
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53. Rewerts JM, McCaw DL, Cohn LA, et  al. Recombinant
human granulocyte colony-stimulating factor for treatment
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54. Mischke R, Barth T, Wohlsein P, et  al. Effect of recombi-
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 CHAPTER 7  Antiviral and Immunomodulatory Drugs
55. Phillips K, Arai M, Tanabe T, et al. FIV-infected cats respond
to short-term rHuG-CSF treatment which results in anti-G-
CSF neutralizing antibody production that inactivates drug
activity. Vet Immunol Immunopathol. 2005;108:357-371.
56. Yamamoto A, Iwata A, Saito T, et al. Expression and puri-
fication of canine granulocyte colony-stimulating factor (cG-
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57. Yamamoto A, Fujino M, Tsuchiya T, et  al. Recombinant
canine granulocyte colony-stimulating factor accelerates
recovery from cyclophosphamide-induced neutropenia in
dogs. Vet Immunol Immunopathol. 2011;142:271-275.
58. Duffy A, Dow S, Ogilvie G, et al. Hematologic improvement
in dogs with parvovirus infection treated with recombinant
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col Ther. 2010;33:352-356.
59. Arai M, Darman J, Lewis A, et al. The use of human hema-
topoietic growth factors (rhGM-CSF and rhEPO) as a
supportive therapy for FIV-infected cats. Vet Immunol Immu-
nopathol. 2000;77:71-92.
60. Mayer P, Werner FJ, Lam C, et al. In vitro and in vivo activity
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ulating factor in dogs. Exp Hematol. 1990;18:1026-1033.
61. Weiss RC, Cox NR, Oostrom-Ram T. Effect of interferon
or Propionibacterium acnes on the course of experimentally
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62. Zeidner NS, Myles MH, Mathiason-DuBard CK, et al. Alpha
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63. Penzo C, Ross M, Muirhead R, et al. Effect of recombinant
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64. Ishida T, Shibanai A, Tanaka S, et  al. Use of recombinant
feline interferon and glucocorticoid in the treatment of feline
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65. Ritz S, Egberink H, Hartmann K. Effect of feline interferon-
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infectious peritonitis. J Vet Intern Med. 2007;21:1193-1197.
66. de Mari K, Maynard L, Sanquer A, et al. Therapeutic effects
of recombinant feline interferon-omega on feline leukemia
virus (FeLV)-infected and FeLV/feline immunodeficiency
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67. Domenech A, Miro G, Collado VM, et al. Use of recombinant
interferon omega in feline retrovirosis: from theory to prac-
tice. Vet Immunol Immunopathol. 2011;143:301-306.
68. Hennet PR, Camy GA, McGahie DM, et  al. Comparative
efficacy of a recombinant feline interferon omega in refrac-
tory cases of calicivirus-positive cats with caudal stomatitis:
a randomised, multi-centre, controlled, double-blind study in
39 cats. J Feline Med Surg. 2011;13:577-587.
69. Haid C, Kaps S, Gonczi E, et  al. Pretreatment with feline
interferon omega and the course of subsequent infection with
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70. Southerden P, Gorrel C. Treatment of a case of refractory
feline chronic gingivostomatitis with feline recombinant inter-
feron omega. J Small Anim Pract. 2007;48:104-106.
71. Gutzwiller ME, Brachelente C, Taglinger K, et al. Feline her-
pes dermatitis treated with interferon omega. Vet Dermatol.
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72. Paltrinieri S, Crippa A, Comerio T, et al. Evaluation of inflam-
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administration. Vet Immunol Immunopathol. 2007;118:68-74.
65
73. Hampel V, Schwarz B, Kempf C, et al. Adjuvant immunother-
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74. Bianco D, Armstrong PJ, Washabau RJ. A prospective, ran-
domized, double-blinded, placebo-controlled study of human
intravenous immunoglobulin for the acute management of
presumptive primary immune-mediated thrombocytopenia in
dogs. J Vet Intern Med. 2009;23:1071-1078.
75. Levy JK, Crawford PC, Collante WR, et al. Use of adult cat
serum to correct failure of passive transfer in kittens. J Am
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76. Day MJ, Horzinek MC, Schultz RD. WSAVA guidelines
for the vaccination of dogs and cats. J Small Anim Pract.
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77. McCann KB, Lee A, Wan J, et al. The effect of bovine lacto-
ferrin and lactoferricin B on the ability of feline calicivirus (a
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78. Beaumont SL, Maggs DJ, Clarke HE. Effects of bovine lacto-
ferrin on in vitro replication of feline herpesvirus. Vet Oph-
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79. Sato R, Inanami O, Tanaka Y, et al. Oral administration of
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80. Kobayashi S, Abe Y, Inanami O, et al. Oral administration of
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82. Breitschwerdt EB, Davidson MG, Hegarty BC, et al. Predniso-
lone at anti-inflammatory or immunosuppressive dosages in
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83. Chang YF, Novosel V, Chang CF, et al. Experimental induc-
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85. Dellinger RP, Levy MM, Carlet JM, et al. Surviving Sepsis Cam-
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86. Sykes JE, Sturges BK, Cannon MS, et al. Clinical signs, imag-
ing features, neuropathology, and outcome in cats and dogs
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87. Baskett A, Barton MH, Norton N, et  al. Effect of pent-
oxifylline, flunixin meglumine, and their combination on a
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1291-1299.
88. Barton MH, Moore JN, Norton N. Effects of pentoxifylline
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89. Marsella R, Olivry T. The ACVD task force on canine atopic
dermatitis (XXII): nonsteroidal anti-inflammatory pharmaco-
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90. Nichols PR, Morris DO, Beale KM. A retrospective study
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2001;12:255-264.
91. Fischer Y, Ritz S, Weber K, et al. Randomized, placebo con-
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Vet Intern Med. 2011;25:1270-1276.
CHAPTER 8

Antibacterial Drugs
Jane E. Sykes and Mark G. Papich

w KEY POINTS
• A
 n understanding of the mechanisms of action, spectrum of • T he importance of appropriate administration of antibiotics,
activity, pharmacokinetics, pharmacodynamics, and adverse compliance issues, and potential adverse effects of the drug
effects of antibacterial drugs facilitates optimal treatment of bac- chosen should be discussed with the pet owner when antibiot-
terial infections while minimizing the chance for (1) selection of ics are prescribed.
resistant bacteria, (2) adverse reactions, and (3) drug interactions.

INTRODUCTION action of β-lactam antibiotics. For example, inhibition of PBP1a

and PBP1b leads to cell lysis, whereas inhibition of PBP2 results
This chapter reviews the classification, mechanisms of action, in rounded cells called spheroblasts. Drugs that produce rapid
spectrum of activity, resistance mechanisms, tissue penetration, lysis (e.g., carbapenems) are the most bactericidal and have
clinical use, and adverse effects of the major classes of antibiot- highest affinity for PBP1.
ics used to treat dogs and cats. Antimicrobials have been grouped Resistance to β-lactam antibiotics is now widespread and
together alphabetically according to their mechanism of action results primarily from β-lactamase production.2 It can also
(cell wall synthesis, nucleic acid, and then protein synthesis inhibi- result from production of altered PBPs (such as PBP2a) and,
tors). Tables are dispersed throughout the text with drug dosages among gram-negative bacteria, exclusion of drugs that normally
for each group of antibiotics. The reader is encouraged to review diffuse through porins to their site of action. Gram-negative
the accompanying text when using the tables. Not all antibiotics β-lactamases are strategically located just beneath the outer
are reviewed here. Antibiotics that have specific uses, such as cer- lipopolysaccharide layer, which acts as the barrier to drug pen-
tain antimycobacterial drugs and nitrofurantoin, are reviewed in etration. Gram-positive bacteria secrete β-lactamases into their
the relevant chapters of this book. Chapter 6 provides an overview immediate surroundings. There are many different β-lactamase
of the basic principles of antimicrobial treatment. enzymes that vary in their specificity for β-lactam drugs.3
β-Lactam antibiotics have a short half-life and exhibit time-
Inhibitors of Cell Wall Synthesis dependent pharmacodynamics (see Chapter 6). Drug concen-
trations should be maintained above the minimum inhibitory
Beta-Lactam Antibiotics concentration (T > MIC) for at least 50% of the dosing interval.
β-Lactam antibiotics have a β-lactam ring in their molecular For penicillins and carbapenems, T > MIC can be less than 50%,
structure (Figure 8-1). They include penicillins, cephalosporins, but aiming for a target of 50% ensures that most patients will
monobactams, and carbapenems. They are bactericidal antibiot- have adequate exposure. To maintain this target, some β-lactam
ics (see Chapter 6) that bind covalently to and inhibit penicillin antibiotics with short half-lives require frequent administration
binding proteins (PBPs). These PBPs are needed to catalyze the or slow infusion. For other drugs, a long half-life prolongs the
cross-linking (or transpeptidation) of the peptidoglycan layer of
bacterial cell walls, which is continuously remodeled by bacteria
(Figure 8-2). When PBPs are inactivated by β-lactam antibiotics,
bacterial enzymes that hydrolyze the peptidoglycan cross-links
during cell wall remodeling continue to function, which breaks
down the cell wall further. The accumulation of peptidoglycan
precursors also triggers activation of cell wall hydrolases, with
further digestion of intact peptidoglycan. The end result is bac-
terial rupture.
The peptidoglycan layer of gram-positive bacteria is 50 to
100 times thicker than that of gram-negative bacteria and is
highly cross-linked, which maintains the structural integrity of
gram-positive bacteria.1 Therefore, compared to gram-negative
bacteria, gram-positive bacteria are more easily inactivated by
β-lactam antibiotics. Bacteria possess multiple PBPs, which are
assigned numbers based on their molecular weight. PBPs vary in
their affinities for different β-lactam antibiotics, which in part
explains the difference in spectrum of activity and bactericidal FIGURE 8-1  Structure of β-lactam antibiotics.

66
 CHAPTER 8  Antibacterial Drugs 67

T > MIC to allow for infrequent administration. For example, 14-day intervals. For other cephalosporins (e.g., injectable drugs
cefpodoxime proxetil has a half-life longer than those of other with short half-lives such as cefotaxime), three- to four-times-
oral cephalosporins, so it can be administered only once per daily dosing may be required for treatment of gram-negative
day. Cefovecin has an extremely long half-life in dogs and cats bacterial infections.
and can be effective for some infections when administered at
Penicillins
Classification, Spectrum of Activity, and Resistance Mechanisms
Penicillins are derived from Penicillium molds. They are clas-
sified as naturally occurring penicillins such as penicillin G
(benzyl penicillin), the semisynthetic aminopenicillins such as
ampicillin and amoxicillin, penicillinase-resistant penicillins,
A and extended-spectrum penicillins. Table 8-1 shows the classifi-
cation of the penicillins and their respective spectra of activity.
Penicillin G must be given parenterally because of poor
oral bioavailability. Gram-positive bacteria that are suscep-
B tible to penicillin G are more susceptible to penicillin G than
aminopenicillins such as ampicillin. The aminopenicillins have
some activity against gram-negative bacteria, although many
gram-negative bacteria are resistant because they produce
β-lactamases. Ampicillin has limited absorption from the gas-
C
trointestinal tract, so it should be given parenterally for treat-
ment of systemic infections. Amoxicillin has approximately
twice the oral absorption of ampicillin and can be given orally.
Penicillinase-resistant penicillins (antistaphylococcal penicil-
D lins) such as methicillin have a structure that resists inactivation
by β-lactamase enzymes. Methicillin is no longer used clinically.
Drugs such as flucloxacillin and dicloxacillin have replaced the
use of methicillin, because they can be administered orally and
E have fewer adverse effects. These drugs are highly protein-bound,
which delays their elimination and maintains plasma drug con-
FIGURE 8-2  Interference of peptidoglycan production by β-lactam antibiotics. The
bacterial cell wall consists of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) centrations, but tissue penetration is sufficient for treatment of
subunits. A, The penicillin binding protein (PBP, brown squares) binds peptide side chains skin and soft-tissue infections. Although dosages of penicillinase-
and forms a cross-link (B). C, The PBP dissociates from the wall once the cross-link has been resistant penicillins have been published for dogs and cats,4 they
formed. D, Penicillins enter the active site of the PBP. E, The β-lactam ring of the penicillin is are rarely used because other alternatives exist such as the com-
irreversibly opened when it reacts with the PBP, which permanently blocks the active site. bination of amoxicillin and the β-lactamase inhibitor clavulanic

TABLE 8-1
Classification and Spectrum of Activity of Penicillin Antibiotics
Penicillin Group Examples Antibacterial Spectrum
Natural penicillins Penicillin G Gram-positive aerobic bacteria (especially streptococci) and anaer-
obes. Pasteurella multocida may also be susceptible.
Aminopenicillins Ampicillin Gram-positive aerobic bacteria (cocci and bacilli) and anaerobes;
Amoxicillin some susceptible gram-negative bacteria.
Penicillinase-resistant Methicillin Susceptible β-lactamase producing gram-positive aerobic bacte-
­penicillins Oxacillin ria. Less active against penicillinase-susceptible organisms than
Cloxacillin penicillin G.
Dicloxacillin
Extended spectrum Ticarcillin Increased activity against susceptible gram-negative aerobes, which
­carboxypenicillins Carbenicillin includes Pseudomonas aeruginosa, Proteus spp., Klebsiella, and
anaerobes. Some reduction in gram-positive spectrum. De-
stroyed by β-lactamases.
Ureidopenicillins Mezlocillin Susceptible gram-positive and gram-negative aerobes, which
Piperacillin includes Pseudomonas aeruginosa. Some loss of anaerobic spec-
trum. Destroyed by β-lactamases.
Penicillin–β-lactamase β-Lactamase inhibitors: Susceptible β-lactamase producing gram-positive and gram negative
inhibitor combinations clavulanic acid, sulbactam, aerobic bacteria and anaerobes.
tazobactam
68 SECTION 2  Antiinfective Therapy

acid. Methicillin and oxacillin are used in the laboratory for sus-
ceptibility testing. Resistance to methicillin results from bacte-
rial production of an altered penicillin-binding protein (PBP2a),
which leads to resistance to all β-lactam antibiotics.5
Extended-spectrum penicillins (antipseudomonal penicil-
lins) have enhanced activity against gram-negative bacteria,
but must be given parenterally. They include the carboxypeni-
cillins, such as ticarcillin and carbenicillin, and piperacillin, an
ureidopenicillin.
Clavulanic acid, sulbactam, and tazobactam are β-lactamase
inhibitors that are administered in conjunction with penicillins,
which increases the spectrum of activity. β-Lactamase inhibitors
possess weak intrinsic antibacterial activity. Clavulanate has
been combined not only with amoxicillin (oral), but also ticar-
cillin (parenteral). Sulbactam has been combined with ampicil-
lin for intravenous (IV) injection. Sulbactam is not as active as
clavulanic acid against some gram-negative β-lactamases. Tazo-
bactam is combined with piperacillin.
Clinical Use
Penicillin dosages for dogs and cats are listed in Table 8-2. Peni-
cillins are distributed to the extracellular fluid of most tissues.
Because of low penetration across the blood-brain barrier and
blood-ocular barrier, high doses are needed in order to attain
effective levels in these tissues. Penicillins undergo rapid renal
elimination, and active drug is concentrated in the urine, so
they are useful for treatment of bacterial urinary tract infections
(UTIs). Liver impairment has little effect on the pharmacokinet-
ics of penicillins. Increasing the length of the dose interval is
important in order to minimize toxicity in animals with renal
failure.
Because of a short half-life, frequent dosing is necessary,
especially when treating gram-negative bacterial infections with
aminopenicillins. Penicillin MICs are generally higher for gram-
negative bacteria than gram-positive bacteria; therefore, higher
penicillin dosages may also be needed for gram-negative bacte-
rial infections.
Adverse Effects
Penicillins are generally well tolerated by dogs and cats. The
most common adverse effects in dogs and cats are vomiting,
diarrhea, and inappetence. These are more likely to occur
with amoxicillin when clavulanic acid is administered concur-
rently. Hypersensitivity reactions that include fever, cutaneous

TABLE 8-2
Suggested Penicillin Dosages for Small Animals
Dose Interval CLSI Breakpoints
Drug (mg/kg) (hours) Species Route Comments (µg/mL)
Penicillin G ­ 20,000-40,000 6-8 D, C IV, IM ≤8 enterococci;
potassium or U/kg ≤0.12 staphylococci
­sodium and streptococci
Ampicillin sodium 10-20 6-8 D, C IV, IM, Administer IV over 3 minutes. ≤0.25 staphylococci,
SC When reconstituted with streptococci, and
sterile water to 250 mg/mL, gram-negative bacilli;
vials should be used within ≤8 canine urinary tract
1 hour. When reconstituted pathogens
with 0.9% NaCl to 45 mg/
mL, stability is maintained
for 48 hours if refrigerated.
Ampicillin-­ 10-20 8 D, C IV, IM See ampicillin. Dose according ≤8/4* staphylococci and
sulbactam to ampicillin component. gram-negative bacteria
Amoxicillin 10-20 8-12 D, C PO See ampicillin.
Amoxicillin-­ 12.5-25 12 D, C PO More frequent or higher ≤0.25/0.12* staphylo-
clavulanate doses recommended for cocci, streptococci,
­potassium ­gram-negative infections. Escherichia coli and
Pasteurella multocida
Ticarcillin ­disodium, 33-50 4-6 D, C IV When reconstituted with ≤64/2* Pseudomonas
­ticarcillin- 0.9% NaCl, stable for up to aeruginosa
clavulanate† 14 days when refrigerated. ≤16/2 gram-negative
enteric bacteria
Piperacillin ­sodium/ 40 6 D, C IV Reconstituted solution should See ticarcillin.
piperacillin-­ be used within 7 days if
tazobactam† ­refrigerated.

C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs.

*The “/” distinguishes the ticarcillin from clavulanic acid concentrations.
†It is recommended that the use of these drugs be reserved for life-threatening infections that are susceptible to these drugs but resistant to other drugs,

on the basis of culture and susceptibility testing.

 CHAPTER 8  Antibacterial Drugs 69

reactions, immune-mediated cytopenias, and anaphylaxis are
possible, especially in dogs, but are less common than in human
patients. The use of penicillins should be avoided in animals
with a history of such reactions. Rapid intravenous infusion
or administration of high doses of penicillins to animals with
coexisting renal failure is rarely followed by development of
neurologic signs, such as seizures. Pain or tissue reactions can
occur when penicillins are administered intramuscularly or sub-
cutaneously. High doses of extended-spectrum penicillins such
as ticarcillin have been associated with platelet function abnor-
malities and significant bleeding in human patients.6
Cephalosporins
Classification and Spectrum of Activity
The cephalosporins were originally derived from Acremonium
spp. (previously known as Cephalosporium spp.). They have
been broadly grouped into first-, second-, third-, and fourth-
generation cephalosporins based on their spectrum of activity.
As the generation increases, there is an increase in gram-negative
spectrum, and fourth-generation drugs have truly broad-spectrum
activity (Table 8-3). An exception to this pattern is the new-
est addition to the group, ceftaroline. It has activity against
­methicillin-resistant staphylococci as a result of an affinity for
PBP2a and has been considered a “fifth-generation” cephalospo-
rin. Cephalosporins are resistant to staphylococcal β-lactamase,
and the extended-spectrum cephalosporins (primarily the
third-generation drugs) are resistant to many gram-negative
β-lactamases. However, extended-spectrum β-lactamase (ESBL)
enzymes can hydrolyze even third-generation cephalosporins
and present an important therapeutic challenge.
Clinical Use
Cephalosporin dosages for cats and dogs are listed in Table 8-4.
First-generation cephalosporins such as cephalexin are moder-
ately absorbed orally and distributed into extracellular fluids. In
general, they have poor penetration of blood-brain barrier, even
in the face of inflammation. Most are excreted unchanged in the
urine, and so are useful for treatment of UTIs. Cefazolin has
greater activity against a wider range of bacterial isolates than
the oral first-generation cephalosporins (e.g., cephalexin and
cefadroxil).7 Cefazolin has been a popular injectable antibiotic
for perioperative use in small animals. This perioperative use of
cefazolin is with the intention to prevent postsurgical infections,
particularly in orthopedic surgery (see Chapter 85).
Use of second-, third-, and fourth-generation cephalosporins
may be indicated when there is resistance to first-generation
cephalosporins and there are no other reasonable alternatives
available. Three third-generation cephalosporins are approved
by the U.S. Food and Drug Administration (FDA) for use in
small animals: ceftiofur, cefpodoxime proxetil, and cefovecin.
These agents are approved for treatment of skin and soft-tissue
infections (cefpodoxime proxetil and cefovecin) and UTIs in
dogs (ceftiofur). Although their use in small animal medicine
has been controversial because of concerns that relate to selec-
tion for resistant bacteria, there is no evidence at this time that
the use of these agents has been associated with increased resis-
tance among isolates from dogs and cats. The injectable third-
generation cephalosporins approved for people have greater in
vitro activity than cefpodoxime or cefovecin and occasionally
have been used in dogs and cats (e.g., cefotaxime, ceftazidime,
cefoperazone). These drugs have a high degree of activity, but
are expensive and must be administered frequently by injec-
tion. Distribution and excretion into urine and bile is variable.
Some third-generation cephalosporins have a longer duration
of activity than first- and second-generation drugs by virtue of
long half-lives. Cefpodoxime proxetil is the most commonly
used oral third-generation drug in small animals, approximately
50% of which is excreted in the urine in active form. Cefove-
cin is approved for use in dogs and cats in Europe and North
America as a subcutaneous injection. The high protein bind-
ing and slow clearance result in a very long half-life in these
species (approximately 7 days in cats and 5 days in dogs).
Administration of cefovecin to cats results in a duration of
activity of at least 14 days, and for at least 7 to 14 days in dogs

TABLE 8-3
Classification and Spectrum of Activity of Cephalosporin Antibiotics
Cephalosporin
­Generation Examples Antibacterial Spectrum
First Cephalexin Methicillin-susceptible staphylococci and streptococci and aerobes; some activity against
Cefazolin gram-negative aerobes (especially cefazolin). Unpredictable activity against anaerobes.
Cefadroxil
Second Cefaclor Improved activity against gram-negative aerobes and anaerobes compared with first-
Cefotetan generation drugs. Cefoxitin has greater stability against anaerobic β-lactamases (such
Cefoxitin as those produced by Bacteroides) than other second-generation drugs.
Third Cefixime Activity primarily against gram-negative bacteria. Some drugs in this group have less
Cefpodoxime activity against gram-positive bacteria and anaerobes.
Ceftiofur
Cefotaxime
Ceftriaxone
Cefovecin
Ceftazidime
Fourth Cefepime Gram-positive cocci, gram-negative bacilli, many β-lactamase-producing gram-negative
bacteria. Reserve for life-threatening infections where no other alternative is available.
70 SECTION 2  Antiinfective Therapy

TABLE 8-4
Suggested Cephalosporin Dosages for Small Animals
Dose Interval
Drug (mg/kg) (hours) Route Species Comments CLSI Breakpoints (µg/mL)
Cephalexin 10-30 6-12 PO D Use 30 mg/kg q12h for pyoderma. ≤2 susceptible organisms.
15-20 12 C More frequent dosing (q6-8h) Cephalothin can be used
is required for gram-negative to test susceptibility for all
bacterial infections. other 1st generation drugs.
Cefadroxil 22 12 PO D See cephalexin.
24 C
Cefazolin sodium 20-35 8 IV, IM D, C 22 mg/kg q2h during surgery See cephalexin.
if indicated.
Cefaclor† 15-20 8 PO D, C ≤8 all susceptible organisms
Cefoxitin sodium† 30 6-8 IM, IV D, C Pain on injection IM. See cefaclor.
Cefixime† 10 12 PO D, C Use 5 mg/kg q12-24h for ≤1 all susceptible organisms
urinary tract infections.
Cefpodoxime proxetil† 5-10 24 PO D* Of all third-generation cephalo- ≤2 all susceptible organisms
sporins, most active against
staphylococci. Less active
against gram-negative and
anaerobic infections.
Ceftiofur sodium† 2.2-4.4 24 SC D* For treatment of otherwise resistant Not established for dogs
urinary tract ­infections. and cats
Cefotaxime sodium† 50 12 IV, IM D Partially metabolized by the liver See cefpodoxime.
20-80 6 C to desacetyl cefotaxime which
contributes to antibacterial
activity. Good CNS penetra-
tion. Stable for 5 days in the
refrigerator after reconstitution.
Ceftazidime† 30 6 IV D, C Potent antipseudomonal ­activity. ≤4 Enterobacteriaceae, ≤8
Pseudomonas aeruginosa
and staphylococci
Cefovecin† 8 14 days SC D, C Approved for use for skin and soft No approved breakpoint.
tissue infections and in some ≤2 all susceptible organ-
countries, urinary tract infec- isms is suggested
tions. Efficacy for treatment
of infections in other sites not
established.
Cefepime† 40 6 IV, IM D* Reduce frequency in dogs with ≤ 8 all susceptible organisms
renal failure (q12-24h).

C, Cats; CLSI, Clinical and Laboratory Standards Institute; CNS, central nervous system; D, dogs.
*Dose not established for cats.
†It is recommended that the use of most second-, third-, and fourth-generation cephalosporins be reserved for serious infections that are susceptible

to those drugs but resistant to other reasonable alternatives on the basis of culture and susceptibility testing.

(depending on the pathogen). Injections can be repeated in dogs
at 14-day intervals if necessary.
Adverse Effects
Adverse effects of cephalosporins in dogs and cats are similar to
those of penicillins, with gastrointestinal signs and hypersensitiv-
ity reactions being most frequent. Although the incidence is low,
cross-sensitivity can occur between penicillins and cephalospo-
rins, so cephalosporins should be avoided or used with caution
in animals that have a history of reactions to penicillin. Inject-
able cephalosporins can cause phlebitis and pain on injection.
Cephalosporins have the potential to cause false-positive results
on test strips that use copper reduction for urine glucose detec-
tion. Certain cephalosporins, such as cefotetan and ceftriaxone,
may exacerbate bleeding tendencies due to vitamin K antago-
nism.8 Reversible bone marrow suppression has been reported
in dogs given high doses of ceftiofur, cefonicid, and cefazedone
long-term.9
Monobactams (Aztreonam)
Aztreonam is a synthetic β-lactam antibiotic that is resistant
to some β-lactamases. It is effective against a wide range of

TABLE 8-5
Suggested Carbapenem Dosages for Small Animals
Dose Interval
Drug (mg/kg) (hours) Species Route
Imipenem-­ 5 6-8 D, C IV, IM, SC
cilastatin
Meropenem 8.5 12 D, C SC
24 12 D, C IV
Carbapenems should be used for serious infections resistant to other reasonable alternatives, on the basis of culture and susceptibility testing.
C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; MIC, minimum inhibitory concentration.
gram-negative bacteria, including Pseudomonas aeruginosa, but
not gram-positive or anaerobic bacteria. In human medicine,
aztreonam is used to treat patients with serious gram-negative
infections who are allergic to penicillins and cannot tolerate
aminoglycosides.10 Dosages have not been established for dogs.
Carbapenems
Spectrum of Activity and Clinical Use
Carbapenems are highly resistant to almost all bacterial
β-lactamases and include imipenem, meropenem, ertapenem,
and doripenem. They penetrate the outer membrane of gram-
negative bacteria more effectively than many other β-lactam
antibiotics and bind to a variety of PBPs, which leads to rapid
lysis of a broad spectrum of bacteria.11 In contrast to other
β-lactam antibiotics, carbapenems have a postantibiotic effect
(see Chapter 6) similar to that of aminoglycosides and fluo-
roquinolones. Although effective against many gram-positive,
gram-negative, and anaerobic bacterial infections, the use
of carbapenems is reserved for treatment of serious, multiple
drug-resistant (MDR) gram-negative infections, especially Esch-
erichia coli, Klebsiella pneumoniae, and P. aeruginosa, that are
resistant to other antibiotics. Methicillin-resistant staphylococci
are resistant to carbapenems. Dosages for dogs and cats are
listed in Table 8-5.
The identification of carbapenemase production by some
strains of gram-negative bacteria has raised great concern,
because this confers resistance to a broad range of β-lactam
antibiotics. Carbapenemase-producing K. pneumoniae (KPC)
are resistant not only to all β-lactam antibiotics, but also to fluo-
roquinolones and aminoglycosides.12 K. pneumoniae that pro-
duce a plasmid-encoded metallo-β-lactamase enzyme (NDM-1)
appeared in India in 2008 and have subsequently spread world-
wide.13 The only options left for treatment of these infections
are tigecycline and polymyxins, both of which have been asso-
ciated with emergence of resistance during treatment.14 The
potential for spread of K. pneumoniae isolates that are resistant
to all commercially available antibiotics now exists. Carapen-
emase (NDM-1) production was recently detected in an E. coli
isolate from a cat in the United States.
CHAPTER 8  Antibacterial Drugs 71
CLSI Breakpoints
Comments (µg/mL)
250 to 500 mg of reconstituted drug ≤1 Enterobac-
should be added to no less than teriaceae, ≤2
100 mL of fluids and administered Pseudomonas
IV over 30-60 minutes. Stable for aeruginosa
24 hours after reconstitution when
refrigerated. For IM injections,
reconstitute with 1% lidocaine.
SC injections may cause alopecia See imipenem.
at the injection site. For Pseudo-
monas aeruginosa or infections
with MIC values approaching the
breakpoint, use 12 mg/kg q8h SC
or 24 mg/kg q8h IV.
Adverse Effects
Imipenem is degraded by dehydropeptidase-1, a brush border
enzyme in the proximal renal tubules, which results in produc-
tion of an inactive metabolite that is nephrotoxic. In order to
prevent nephrotoxicity and maximize imipenem’s antibacterial
activity, imipenem is administered with cilastatin, which inhib-
its the dehydropeptidase-1 enzyme. Newer carbapenems such as
meropenem are resistant to dehydropeptidase-1.
Adverse effects of carbapenems are otherwise rare and
include vomiting, nausea, and pain on injection. Neurologic
signs, including tremors, nystagmus, and seizures, can occur
following rapid infusion of imipenem-cilastatin or in animals
with renal insufficiency. Imipenem must be administered slowly
in intravenous fluids (see Table 8-5). Slow administration is not
required for meropenem, which is more soluble. Subcutaneous
injections of meropenem have produced alopecia at the injec-
tion site,4 but otherwise these injections have been very well
tolerated and have eradicated infections associated with MDR
gram-negative bacteria.
Glycopeptides
Mechanism of Action and Clinical Use
Glycopeptides are cyclic glycosylated peptide antimicrobials
that inhibit the synthesis of peptidoglycan by binding to amino
acids (d-alanyl-d-alanine) in the cell wall, preventing the addi-
tion of new units. Vancomycin, teicoplanin, and decaplanin are
all glycopeptides, but only vancomycin has been used in small
animals.
Vancomycin was discovered in the 1950s, but has not been
frequently used in small animals because it is not absorbed
orally, so IV infusion is required for treatment of systemic
infections. When administered IV, it can be valuable for treat-
ment of MDR gram-positive bacterial infections, such as
methicillin-resistant staphylococcal infections, resistant entero-
coccal infections, and encrusting cystitis caused by Corynebac-
terium urealyticum. The last is an emerging infection of dogs,
cats, and immunocompromised human patients, treatment of
which requires surgical debridement of the bladder in conjunc-
tion with glycopeptide administration.15,16 Vancomycin is a
72 SECTION 2  Antiinfective Therapy
valuable agent for treatment of methicillin-resistant staphylo-
cocci, because resistance among these organisms is extremely
rare, and the bactericidal activity can be an advantage when
treating immunocompromised patients. In human patients, oral
vancomycin has been used to treat gastrointestinal infections
caused by resistant Clostridium difficile.
Vancomycin-resistant methicillin-resistant Staphylococcus
aureus (MRSA) isolates are extremely rare in people and have
only been described in specific geographic locations. Vancomycin-
resistant Staphylococcus pseudintermedius isolates have not yet
been described. Vancomycin-resistant enterococci (VRE) are
well documented in human patients and can present a therapeutic
challenge. Although currently rare, the existence of VRE has
been reported in companion animals.17 Resistance to vancomycin
results from bacterial alteration of the terminal amino acid to
which vancomycin binds. In order to reduce inappropriate use
of vancomycin, some laboratories do not report results for van-
comycin susceptibility unless reporting is indicated on the basis
of resistance in the remainder of the susceptibility panel.
Teicoplanin has a longer half-life and is up to 100 times
more lipophilic than vancomycin. It is not commercially avail-
able in the United States but is used in Europe. Vancomycin
and teicoplanin appear to be equally efficacious in human
patients.18
Adverse Effects
Vancomycin is not commonly used to treat small animals,
and so adverse reactions have not been well documented. The
most common reaction in human patients is the “red man syn-
drome” caused by histamine release after rapid infusion. This
can be prevented through the use of antihistamines and slower
infusion rates. Other adverse reactions are rare. Previously
cited nephrotoxicity from vancomycin may be exaggerated
because of its frequent co-administration with aminoglyco-
sides. Current formulations are of better quality and purity and
less likely to produce kidney injury. Vancomycin is given by
IV infusion over 30 to 60 minutes through a central or periph-
eral catheter (Table 8-6). Trough plasma concentrations can be
monitored and should be greater than 10 µg/mL for skin and
soft tissue infections, and 10 to 20 µg/mL for more compli-
cated infections. Intramuscular administration is painful and
irritates tissues.
TABLE 8-6
Suggested Vancomycin Dosages for Small Animals
Drug Dose (mg/kg) Interval (hours) Species Route Comments
Vancomycin* 15 6-8 D IV
12-15 8 C
C, Cats; CLSI, Clinical and Laboratory Standards Institute; CRI, continuous-rate infusion; D, dogs.
*The use of vancomycin should be reserved for gram-positive infections that are susceptible to vancomycin but resistant to all other reasonable
alternatives, on the basis of culture and susceptibility testing.
Nucleic Acid Inhibitors
Fluoroquinolones
Classification and Mechanism of Action
The fluoroquinolones bind to DNA gyrase (also known as topoi-
somerase II) and topoisomerase IV, enzymes that cleave DNA
during DNA replication. The result is disruption of bacterial
DNA and protein synthesis. For the veterinary fluoroquinolones
enrofloxacin, orbifloxacin, and marbofloxacin, DNA gyrase is
the primary target for gram-negative bacteria and topoisomer-
ase IV is the primary target for gram-positive bacteria. Because
topoisomerase IV has a lower affinity than DNA gyrase for this
group of drugs, higher MICs are observed for gram-positive
bacteria compared to the Enterobacteriaceae.
Fluoroquinolones were created by addition of a fluorine group
to nalidixic acid, which improves its spectrum of activity. Exam-
ples are enrofloxacin, ciprofloxacin, difloxacin, orbifloxacin, and
marbofloxacin. Newer-generation fluoroquinolones inhibit both
DNA gyrase and topoisomerase IV in gram-positive bacteria,
leading to enhanced activity for treatment of gram-positive and
anaerobic bacterial infections and reduced likelihood of selection
for resistant mutants. Pradofloxacin, levofloxacin, moxifloxa-
cin, and gatifloxacin are third-generation fluoroquinolones. The
only antibiotic in this group approved for use in companion ani-
mals is pradofloxacin, which is approved for use in cats in the
United States, and approved for both dogs and cats in European
countries.
Resistance to fluoroquinolones results from DNA gyrase
mutations, decreased bacterial permeability, and increased
drug efflux.19 Susceptibility to one fluoroquinolone generally
predicts susceptibility to others, with the exceptions of third-
generation fluoroquinolones (such as pradofloxacin) and cipro-
floxacin, which has higher in vitro activity against P. aeruginosa
than other fluoroquinolones.20,21 Limited use of fluoroquino-
lones has been recommended by some human and veterinary
medical authorities in order to minimize further development
of resistance, but a clear association between prescription of
fluoroquinolones and increased resistance has not been estab-
lished in companion animals. Nevertheless, other “first line”
drugs are often available for most infections, and the valuable
fluoroquinolones can be reserved for cases that cannot tolerate
other drugs, or when resistance to other drugs is more likely.
CLSI Breakpoints
(µg/mL)
Reconstituted drug is added to 0.9% ≤4 Enterococcus and
NaCl or 5% dextrose and admin- Staphylococcus
istered over 30 to 60 minutes.
It can also be administered in 5%
dextrose as a CRI, with a loading
dose of 3.5 mg/kg, and then
1.5 mg/kg/hr IV. Therapeutic
drug monitoring recommended.
 CHAPTER 8  Antibacterial Drugs 73

A sufficiently high dose should be used to minimize selection of
resistant isolates.
Clinical Use
Fluoroquinolones are most valuable to treat gram-negative infec-
tions, because there are often few oral medication alternatives
for these infections. The fluoroquinolones are also effective for
infections caused by staphylococci, but staphylococci tend to have
higher MICs for fluoroquinolones than gram-negative bacteria.
The activity of fluoroquinolones against anaerobes is variable.
Pradofloxacin has the greatest activity, followed by marboflox-
acin and enrofloxacin.22 Fluoroquinolones are concentration-
dependent antibiotics, so they are best administered as a single
high daily dose. Doses for fluoroquinolones are listed in Table 8-7.
Fluoroquinolones are usually well absorbed after oral admin-
istration. Poor absorption occurs when they are complexed
by divalent and trivalent cation-containing medications (e.g.,
antacids) and supplements (aluminum, calcium, iron, zinc).
Use of these should be avoided if animals are receiving fluo-
roquinolones by the oral route. Oral absorption of the human
drug ciprofloxacin is lower and more variable in dogs than
in people, which necessitates administration of a higher dose.
In one study, oral absorption of generic ciprofloxacin tablets in
dogs was 58%, but the coefficient of variation was 45%.23 In
cats, only 22% to 33% of orally administered ciprofloxacin is
absorbed. Most fluoroquinolones are highly concentrated in the
urine, so they are useful for treatment of UTIs. Enrofloxacin
is metabolized to ciprofloxacin, which is subsequently excreted
in the urine. Approximately half of the administered dose of
marbofloxacin and orbifloxacin is excreted as unchanged drug
in the urine. In contrast, difloxacin is excreted primarily in bile,
and little drug enters the urine.24

TABLE 8-7
Suggested Fluoroquinolone Dosages for Small Animals
Dose Interval
Drug (mg/kg) (hours) Species Route Comments CLSI Breakpoints (µg/mL)
Enrofloxacin 5-20 24 D IV, IM, PO Interactions can occur with certain ≤0.5 susceptible infections.
5 24 C* IM, PO oral medications (see text). Use higher dosages (10-20
Reduce dose or increase dosage mg/kg) for organisms with
interval with renal failure. Dilute MICs of 1 or 2 (except in
in fluids (e.g., 1:10 dilution) and cats). A dose of 5 mg/kg
infuse IV over 30 minutes. may be sufficient for UTIs.
Ciprofloxacin 20-30 24 D, C PO See enrofloxacin. Oral ­absorption In humans, ≤1 susceptible
­hydrochloride 10 24 D, C IV may be limited (see text). infections. More active
against Pseudomonas
aeruginosa than other
fluoroquinolones
Marbofloxacin 2.75-5.5 24 D, C PO See enrofloxacin. ≤1 susceptible infections. Use
higher doses for bacteria
with higher MICs.
Orbifloxacin 2.5-7.5 24 D, C PO See enrofloxacin. Orbifloxacin See marbofloxacin.
suspension provides lower and
more variable plasma levels than
the tablets. A dose of 7.5 mg/kg
should be used.
Difloxacin 5-10 24 D PO Urine concentrations may not be ≤0.5 susceptible ­infections
­hydrochloride sufficient for treating UTIs.
Moxifloxacin 10 24 D, C PO Moxifloxacin has enhanced activi- See marbofloxacin.
ty against gram-­positive bacteria
and anaerobes. In humans, urine
concentrations are not sufficient
for treatment of UTIs.
Pradofloxacin 3† 24 D, C PO See moxifloxacin for spectrum of Not established
5-7.5‡ C activity. Food reduces bioavail-
ability. Appears effective for
treatment of UTIs.

C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; MIC, minimum inhibitory concentration; UTI, urinary tract infection.
*The use of enrofloxacin to treat cats is controversial because of the potential for irreversible retinal toxicity and blindness. Do not exceed a dose
of 5 mg/kg in cats. The use of alternative fluoroquinolones such as marbofloxacin, orbifloxacin, or pradofloxacin should be considered for cats.
†Tablet formulation. The use of pradofloxacin at high doses in dogs has been associated with myelosuppression and is off-label in the United States,

but it is approved for both dogs and cats outside the United States at a dose of 3 mg/kg (tablets).
‡Oral suspension for cats. The dose of the oral suspension for cats is higher (5-7.5 mg/kg) owing to reduced bioavailability.
74 SECTION 2  Antiinfective Therapy
Fluoroquinolones have variable protein binding, but this does
not inhibit distribution to tissue fluids. Because of good lipophilic-
ity, these drugs penetrate the prostate and respiratory secretions.
They also penetrate the ear canal, but high doses may be required
for treatment of bacteria with relatively high MIC values.25 Fluo-
roquinolones can attain high intracellular concentrations and can
be used to treat infections caused by intracellular pathogens such
as Mycoplasma spp. and some Mycobacterium spp.
Adverse Effects
Fluoroquinolones can occasionally produce gastrointestinal
signs in dogs and cats, such as decreased appetite and vomit-
ing. Rapid IV administration can cause systemic hypotension,
tachycardia, and cutaneous erythema, possibly as a result of
histamine release.26 Neurologic signs, including tremors, ataxia,
and seizures, can occur in dogs and cats treated with high doses
of parenteral fluoroquinolones. Although not well understood,
this is thought to result from fluoroquinolone interference with
the binding of γ-aminobutyric acid to its receptors in the central
nervous system (CNS).27 In general, fluoroquinolones should
be used cautiously in animals prone to seizures. Enrofloxacin
causes hallucinations in humans and so is not marketed or
approved for treatment of humans.
Cats treated with high doses of enrofloxacin (generally >5
mg/kg/day), especially parenteral enrofloxacin, have developed
blindness resulting from acute retinal degeneration, manifested as
bilateral mydriasis with tapetal hyperreflectivity.28,29 This results
from a functional defect in a fluoroquinolone transport protein
in the cat, with subsequent accumulation of photoreactive drug
in the retina.30 In general, the blindness is irreversible, but if it is
detected early and medication is discontinued, some resolution
may occur. Orbifloxacin also can produce retinal injury in cats,
but the dose needed for this reaction is much higher than doses
used therapeutically. Studies of marbofloxacin (reported by the
manufacturer) and pradofloxacin31 showed no evidence of retinal
toxicity in cats, even with high doses, although there are reports
to the FDA of blindness in cats associated with the use of mar-
bofloxacin. Cats with renal insufficiency or those treated with
medications that increase plasma fluoroquinolone concentrations
may be more likely to develop this adverse effect.
Because they inhibit proteoglycan synthesis and chelate mag-
nesium, fluoroquinolones can cause cartilage and joint toxic-
ity in young animals, although the clinical importance of this
has been questioned.32,33 In general, prolonged (>7 days) use
TABLE 8-8
Suggested Metronidazole Dosages for Small Animals
Drug Dose (mg/kg) Interval (hours) Species
Metronidazole 15 12 D PO
10-15 24 C dysfunction. An intravenous solution, metroni-
Metronidazole 25 12 C PO
benzoate
C, Cats; D, dogs.
of fluoroquinolones during the period of rapid growth in dogs
(from about 1 to 2 months of age to 28 weeks) should be avoided
if possible. Cats are thought to be more resistant to development
of cartilage toxicity. Pradofloxacin has been associated with
myelosuppression in dogs at doses greater than 10 mg/kg.
Fluoroquinolones inhibit some cytochrome P450 enzymes,
which decreases metabolism of other drugs. This occurs with
theophylline in dogs; therefore, the dose of theophylline should
be adjusted if administered concurrently. Like cephalosporins,
fluoroquinolones can cause false-positive results on test strips
for urine glucose detection. Cutaneous photosensitization
occurs in human patients but is rare in animals. Other adverse
effects reported in humans, but not in dogs and cats, include
cardiac arrhythmias, tendinitis and tendon rupture, altered glu-
cose metabolism, and hepatic dysfunction.34
Metronidazole
Mechanism of Action and Spectrum of Activity
Metronidazole is a bactericidal nitroimidazole drug that is used
primarily to treat anaerobic bacterial infections (both gram-
negative and gram-positive), which includes those caused by
Bacteroides fragilis and Clostridium spp., and infections with
certain protozoa, such as Giardia spp. Metronidazole diffuses
into bacterial cells as a prodrug and is activated in the cyto-
plasm. Once within the cell, the nitro group of metronidazole
preferentially accepts electrons from electron transport proteins
such as ferredoxin. A short-lived nitroso free radical is thus gen-
erated that damages DNA. The intermediate compounds then
decompose into non-toxic, inactive end products.
Metronidazole has antioxidant properties that are believed
to result from its ability to scavenge reactive oxygen species and
modulation of neutrophil activity.35 It is often used at low doses
to treat inflammatory bowel disease in dogs and cats,36 but it
is not known if the benefit is from the antibacterial activity or
direct effects on inflammatory cells.
Resistance to metronidazole is rare among anaerobes.37
Mechanisms of resistance include reduced drug uptake and
decreased reduction activity. Other proposed mechanisms
include increased efflux, drug inactivation, and accelerated
DNA repair.
Clinical Use
In dogs and cats, metronidazole is used to treat anaerobic bacte-
rial and protozoal infections in a variety of tissues. Suggested
Route Comments
Reduce dose by 50% in animals with hepatic
dazole hydrochloride, is available but is highly
acidic. It should be diluted in 100 mL of IV
crystalloids and neutralized with 5 mEq sodium
bicarbonate per 500 mg for a pH of 6-7. Di-
luted solutions are stable for 24 hours.
More palatable formulation for cats may be
available from some compounding pharmacies.

doses are listed in Table 8-8. Metronidazole is rapidly and almost
completely absorbed from the gastrointestinal tract, diffuses
across lipid membranes, and achieves therapeutic concentrations
in tissue fluids. Metronidazole is metabolized by the liver. The
metabolites, along with intact drug, are excreted in the urine.
Adverse Effects
The main adverse effect of metronidazole is neurotoxicity, which
tends to occur with high doses (>30 mg/kg/day) or in animals
with hepatic dysfunction. Clinical signs of neurotoxicity are
reversible on discontinuation of the drug and include lethargy,
truncal ataxia, hypermetria, intention tremors, head tilt, falling,
vertical nystagmus, extensor rigidity, opisthotonos and seizures,
and reflect damage to the vestibular system.38,39 Although not
fully understood, neurotoxicity is believed to result from antag-
onism of γ-aminobutyric acid in the cerebellar and vestibular
systems. Neurologic signs resolve more quickly when treated
with a low dose of diazepam (0.4 mg/kg PO q8h for 3 days).38
Bilateral symmetrical cerebellar lesions, most commonly in the
dentate nucleus have been reported on magnetic resonance
imaging in human patients with metronidazole toxicity,40 and
multifocal necrotic foci were detected at necropsy in the brain-
stem of a cat.39 Administration of metronidazole to animals
with a history of metronidazole toxicosis is not recommended.38
Less commonly, metronidazole causes decreased appetite,
vomiting, and diarrhea. Metronidazole suspension has a bitter
taste that may be unpalatable to cats, which can be overcome
by administration of the drug in gel capsules. Although not
approved by the FDA, metronidazole benzoate is a more pal-
atable formulation for cats and pharmacokinetic studies have
demonstrated good absorption.41 There have been concerns
that relate to toxicity from the benzoic acid component of the
formulation, which can cause CNS signs, blindness, and respi-
ratory problems at high doses. However, this possibility is con-
sidered to be unlikely.41 Because metronidazole benzoate is only
62% metronidazole, dosage adjustment is required when this
formulation is used. Metronidazole inhibits hepatic microsomal
enzymes and so may increase the concentration of drugs such as
warfarin and cyclosporine. It also lowers serum lipid concentra-
tions (including cholesterol concentrations) in human patients.42
Rifamycins
Mechanism of Action and Spectrum of Activity
Named after the French crime movie Rififi, rifamycins are
bactericidal antimicrobials that inhibit the β-subunit of
TABLE 8-9
Suggested Rifampin Dose for Small Animals
Interval
Drug Dose (mg/kg) (hours) Species Route Comments
Rifampin 5 12 D, C PO
C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; MIC, minimum inhibitory concentration.
CHAPTER 8  Antibacterial Drugs 75
DNA-dependent RNA polymerase in bacteria. This leads to
impaired RNA synthesis. Only the semisynthetic drug rifampin
(also known as rifampicin) has been used to any great extent in
small animal medicine. Rifampin is active against staphylococci,
streptococci, and only a few gram-negative organisms. When
used as a component of antimycobacterial treatment regimes, it
increases the chance of treatment success. Rifabutin is effective
for treatment of some mycobacterial infections that are resistant
to rifampicin.43
It has been recommended that rifampin be used in combina-
tion with other antimicrobials to reduce the risk of emergence
of rifampin resistance, which occurs quickly when rifampin
is used alone. However, this applies primarily when it is used
for long-term treatment, such as for mycobacterial infections.
Monotherapy has been successful when rifampin has been used
to treat methicillin-resistant Staphylococcus infections, which
accounts for most of the current small animal use. If combina-
tion treatment is pursued for treatment of methicillin-resistant
staphylococci, there are often few effective alternatives with
which to combine rifampin, because these infections are typi-
cally multidrug resistant. Resistance to rifampin results from a
single mutation that leads to an altered RNA polymerase that
does not effectively bind rifampin.
Clinical Use
One of the major advantages of rifampin is its high degree of
lipid solubility, which provides the degree of intracellular pen-
etration required for treatment of infections caused by intracel-
lular bacteria such as Mycobacterium spp. and Brucella canis
infections. Rifampin has been used to treat bartonellosis in com-
bination with doxycycline.44,45 Because of its activity against
staphylococci and the fact that it can be administered orally, it
has been used for treatment of methicillin-resistant staphylococ-
cal infections in small animals. For other infections, rifampin is
less attractive because of its adverse effect profile.
Rifampin dosing is shown in Table 8-9. Rifampin is rapidly
absorbed from the gastrointestinal tract and metabolized by
the liver. Active metabolites are excreted in bile and to a lesser
extent the urine.
Adverse Effects
Vomiting and anorexia are common in dogs treated with
rifampin. The drug can impart a red-orange color to the urine
and, to a lesser extent, tears, saliva, the sclera, and mucous
membranes. Rifampin can also be associated with increased
CLSI Breakpoints
(µg/mL)
Preferably use in combination with other ≤1 for susceptible
drugs. Avoid in animals with hepatic organisms (usual-
dysfunction. Do not administer with fatty ly MICs of gram-
meals. Drug interactions may occur (see positive bacteria
text). Monitor liver enzymes with prolonged fall below this
treatment. May impart a red-orange color breakpoint)
to the urine and tears.
76 SECTION 2  Antiinfective Therapy
serum liver enzyme activities and hepatopathy. Doses that
exceed 10 mg/kg per day in dogs increase the risk of liver injury.
In humans, fever, cutaneous reactions, thrombocytopenia, acute
intravascular hemolytic anemia, and acute tubulointerstitial
nephritis have been reported, which appear to have an immune-
mediated pathogenesis.46 Rarely, pancreatitis has been reported
in human patients receiving rifampin. Adverse effects have not
been reported in cats, but some of these risks may apply to cats
as well as dogs. Because of poor palatability, it is a challenge to
administer to cats.
Rifampin is one of the most potent inducers of hepatic
microsomal enzymes and efflux proteins, such as P-glycoprotein.
Subsequently, it can increase the clearance of several co-­
administered drugs (thereby decreasing their efficacy), such
as glucocorticoids, cyclosporin, itraconazole, and digoxin. In
human patients, recovery from the effect of rifampin on hepatic
microsomal enzymes can take 4 weeks.47
Trimethoprim-Sulfonamides
Mechanism of Action
Trimethoprim and sulfonamides act synergistically to inhibit
folic acid metabolism by bacteria. The combination interferes
with purine and therefore DNA synthesis. Trimethoprim inhib-
its bacterial dihydrofolate reductase. Sulfonamides are chemical
analogs of para-aminobenzoic acid (PABA), and competitively
inhibit the incorporation of PABA into dihydropteroic acid by
the enzyme dihydropteroate synthetase, which also leads to
decreased folic acid synthesis. When administered separately,
each drug is bacteriostatic, but the combination is bactericidal.
Preparations used to treat dogs and cats include a 1:5 ratio of
trimethoprim to sulfonamide. Because folic acid is of dietary
origin in animals, only bacterial purine synthesis is affected.
Resistance to trimethoprim-sulfonamide (TMS) antibiotics
most commonly results from plasmid-mediated production of
altered dihydrofolate reductase or dihydropteroate synthetase,
with reduced binding affinities. Other mechanisms include over-
production of dihydrofolate reductase or PABA by bacteria,
and reduced bacterial permeability to trimethoprim and sulfon-
amides. Multiple mechanisms may operate simultaneously.48
TABLE 8-10
Suggested Trimethoprim-Sulfonamide Dosages for Small Animals
Dose Interval
Drug (mg/kg) (hours) Species
Trimethoprim- 30* 12 D, C
sulfamethoxazole,
trimethoprim-­
sulfadiazine
Ormetoprim- 55 on first day, 24 D PO
sulfadimethoxine and then 27.5*
C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; TMS, trimethoprim-sulfonamide.
*Dose listed is for combined components, (e.g., 5 mg of trimethoprim and 25 mg of sulfonamide).
Clinical Use
TMS antibiotics have a broad spectrum of activity and can be
used to treat gram-positive and many gram-negative bacterial
infections, as well as some protozoal infections. Enterococci
are intrinsically resistant to TMS antibiotics. Many anaerobic
bacteria are not treated successfully because despite in  vitro
susceptibility, the drugs are less active in tissues where anaer-
obes proliferate. TMS antibiotics are the treatment of choice for
nocardiosis. They have also been used successfully for preven-
tion of bacterial infections in dogs being treated with myelosup-
pressive chemotherapeutics such as doxorubicin.49 Prophylactic
protocols have selected for resistant bacteria in humans,50 but
this has not been reported in animals. TMS combinations are
available as either trimethoprim-sulfadiazine or trimethoprim-
sulfamethoxazole (the latter is a human medical preparation),
which are used interchangeably. However, the veterinary
formulation of trimethoprim-sulfadiazine is rarely available
commercially, and most veterinarians rely on trimethoprim-
sulfamethoxazole. No difference in activity between the two
formulations has been documented, but this has never been eval-
uated. The primary difference between the formulations is that
sulfadiazine is more water soluble and is excreted in the urine in
an unchanged form. A similar formulation to the trimethoprim-
sulfonamides for use in veterinary medicine is ormetoprim sul-
fadimethoxine, which has been used for the same indications as
trimethoprim-sulfonamides. Doses for TMS combinations are
listed in Table 8-10.
TMS is well absorbed after oral administration. As a weak
base, trimethoprim is able to penetrate the blood-prostate bar-
rier and can be used to treat prostatic infections. Both trim-
ethoprim and sulfonamides are metabolized to some extent by
the liver. Both active drug and inactive metabolites appear in
the urine. Active TMS is highly concentrated in urine and is an
appropriate first choice for treatment of UTIs in dogs and cats.51
Adverse Effects
TMS antimicrobials cause a wide range of adverse effects, which
have somewhat limited their use. These reactions are primar-
ily caused by the sulfonamide component. Some of the adverse
CLSI Breakpoints
Route Comments (µg/mL)
PO, IV Avoid in animals with hepatic ≤2/38 for suscep-
failure. Reduce dose in animals tible infections
with renal insufficiency. Obtain
baseline Schirmer tear test and
monitor this and the CBC with
prolonged treatment. Each 5-mL
vial of the injectable preparation
should be diluted in 75-125 mL
of 5% dextrose and adminis-
tered IV over 1 hour.
See trimethoprim-­ Use information
sulfamethoxazole. for TMS.

effects may be attributed to the inability of dogs to acetylate
sulfonamide compounds. People who are “slow acetylators” are
also more prone to adverse effects from sulfonamides.
Gastrointestinal signs such as vomiting can occur. Other
common adverse effects in dogs are hypersensitivity reactions
and include keratoconjunctivitis sicca, fever, polyarthritis,
cutaneous drug eruptions,52,53 immune-mediated thrombocy-
topenia, hemolytic anemia, hepatitis, pancreatitis, meningitis,
interstitial nephritis, glomerulonephritis, and aplastic anemia­
(Figure 8-3).54-57 These events are relatively uncommon, but
have been documented well enough in dogs that it has discour-
aged their routine use among some veterinary clinicians. Revers-
ible hypothyroidism can occur after periods of treatment as
short as 10 days.58 Hepatic necrosis can occur as an idiosyncratic
reaction.59 Ormetoprim has been associated with neurologic
signs in dogs, including tremors, anxiety, and seizures. Schirmer
tear tests should be performed before sulfonamides are admin-
istered to dogs, tear tests should be monitored when prolonged
treatment is required (e.g., greater than 1 week), and the drug
should be avoided in dogs with keratoconjunctivitis sicca. Sul-
fonamide cystolithiasis can occur in dogs treated with high doses
of sulfonamides.60 Doberman pinschers may be more susceptible
than other breeds to adverse effects of TMS because of their
inability to detoxify certain sulfonamide metabolites; in another
study, Samoyeds and miniature schnauzers also appeared to be
more susceptible.55,61
Protein Synthesis Inhibitors
Aminoglycosides
Mechanism of Action
All aminoglycosides contain an essential six-membered ring with
amino-group substituents and are highly positively charged.
Aminoglycosides with names that end in -mycin derive directly
or indirectly from Streptomyces spp. (e.g., neomycin). Those
with names ending in -micin are derived from Micromonospora
(e.g., gentamicin).
Aminoglycosides have multiple mechanisms of action. They
bind electrostatically to the bacterial outer membrane and dis-
place cell wall Mg2+ and Ca2+, which normally link adjacent
lipopolysaccharide molecules. This initial binding is greater in
FIGURE 8-3  Right eye of a 7-year-old intact male golden retriever that developed ker-
atoconjunctivitis sicca after administration of trimethoprim-sulfamethoxazole for 3 weeks
for septic peritonitis. The owner noticed the dog pawing at his eyes. Mucoid ocular discharge
is present at the medial canthus. Schirmer tear test strip results were 0 mm for both eyes.
(Image courtesy University of California, Davis Veterinary Ophthalmology service.)
CHAPTER 8  Antibacterial Drugs 77
gram-negative bacteria than gram-positive bacteria because of a
greater presence of an outer membrane in gram-negative organ-
isms. The end result is disrupted cell permeability. Subsequently,
the bacterial cell takes up the aminoglycoside molecules, and
they become trapped irreversibly within the cytoplasm. Because
this is an oxygen-dependent process, anaerobes are intrinsically
resistant to aminoglycosides. Once within the cell, aminoglyco-
sides bind to the 30S subunit of the bacterial ribosome, which
results in decreased protein synthesis.
Aminoglycosides, particularly gentamicin, amikacin, and
tobramycin, are highly active compounds for which resistance
is uncommon. The most common mechanism of aminoglyco-
side resistance is enzymatic modification of the drug by bacte-
ria. More than 50 modifying enzymes have been identified.62
Reduced drug uptake by bacteria may also occur. Enterococci
are intrinsically resistant to low levels (4 to 250 µg/mL) of ami-
noglycosides because of their anaerobic metabolism, and thus
they require testing for high-level resistance using higher concen-
trations of aminoglycosides. Resistance to one aminoglycoside
does not imply resistance to others. For example, tobramycin
and amikacin are consistently more active than gentamicin, and
kanamycin is one of the least active aminoglycosides. Because of
these differences, a susceptibility panel should include gentami-
cin and either amikacin or tobramycin.
Clinical Use
Aminoglycosides are primarily used to treat gram-negative
bacterial infections and MDR staphylococcal infections. They
should not be used to treat anaerobes, and Streptococcus species
are better treated with β-lactams. Aminoglycosides have usu-
ally been administered together with cell wall–active antimicro-
bial drugs (β-lactams and glycopeptides), but this is no longer
believed to be a therapeutic advantage. They are very water sol-
uble and poorly lipid soluble, so they penetrate tissue fluids well,
but do not readily penetrate tissues that are dependent on lipid
diffusion (prostate, brain, eye, or cerebrospinal fluid). Penetra-
tion of bile is also poor. Oral absorption is negligible, and they
should not be administered orally unless specifically intended
to treat intestinal bacteria (e.g., oral neomycin for treatment of
hepatic encephalopathy). Doses are shown in Table 8-11. Ami-
noglycosides also may be administered topically. Because they
penetrate bronchial secretions poorly, nebulization has been
used in order to overcome this problem in human patients as
well as dogs with resistant gram-negative bacterial broncho-
pneumonia (see Table 8-11).63 Aminoglycosides are excreted
unchanged by the kidney and concentrate 25- to 100-fold in
urine within an hour of drug administration, so they can be used
to treat resistant gram-negative bacterial UTIs. They also accu-
mulate in renal tissue and remain active for several days after
dosing is discontinued. Because they are not well distributed
into fat, dose reduction may be indicated for obese animals.
Despite their primary mechanism of action being protein
synthesis inhibition, aminoglycosides are rapidly bactericidal
with a prolonged, concentration-dependent postantibiotic
effect. Transient high peak drug concentrations result in faster
killing and greater bactericidal activity than lower concentra-
tions over a prolonged period of time (i.e., they are concentra-
tion-dependent antimicrobial drugs). Once-daily dosing of a
high dose of an aminoglycoside is therefore preferred because
it achieves optimal bacterial killing but minimizes toxicity. In
order to further minimize toxicity, aminoglycosides are gener-
ally infused slowly. In human patients, it is recommended they
78 SECTION 2  Antiinfective Therapy

TABLE 8-11
Suggested Aminoglycoside Dosages for Small Animals
Dose Interval CLSI Breakpoints
Drug (mg/kg) (hours)* Species Route Comments (µg/mL)
Gentamicin 9-14 24 D IV, IM, SC Ensure adequate fluid and electrolyte ≤2 susceptible
sulfate 5-8 24 C balance during treatment. Avoid infections
in animals with renal impairment.
Monitor BUN and creatinine.
Amikacin and tobramycin are more
active against some gram-negative
bacteria than gentamicin, but
gentamicin is preferred for Serratia
infections. Do not mix in vials with
other drugs.
Amikacin 15-30 24 D IV, IM, SC See gentamicin. ≤16 susceptible
10-14 24 C infections
Tobramycin 9-14 24 D IV, IM See gentamicin. For nebulization, 160 ≤4 susceptible
sulfate 5-8 24 C mg of nebulization solution† q12h infections
for 28 days. Preferred aminoglyco-
side for Pseudomonas infections.
Kanamycin 20 24 D, C IV or IM See gentamicin. Less active than genta- ≤16 susceptible
sulfate micin and amikacin. infections
Neomycin 10-20 6-12 D, C PO Not absorbed systemically; used to Not applicable
treat hepatic encephalopathy. Use
cautiously in animals with renal
disease and avoid use for >14 days.

C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; MIC, minimum inhibitory concentration.
*Once-daily administration is preferred except for enterococcal endocarditis, where the continuous presence of both an aminoglycoside and a penicil-
lin is recommended (see Chapter 86).
†TOBI nebulization solution. Injectable tobramycin or gentamicin can be used but may be associated with bronchospasm. They should be diluted in

3 mL of sterile saline, and albuterol should be administered just before nebulization.

be administered over a 15- to 30-minute period. Subcutaneous
and intramuscular injections of water-soluble formulations of
aminoglycosides are also acceptable routes of administration,
although they may cause pain in some animals.
Adverse Effects
Aminoglycosides have the potential to cause nephrotoxicity,
ototoxicity, and neuromuscular blockade. Their cationic struc-
ture and the high anionic phospholipid concentrations in the
kidney and inner ear play an important role in toxicity. In the
presence of gastrointestinal ulceration, systemic absorption of
orally administered aminoglycosides can occur, with associated
nephrotoxicity or ototoxicity.64
Nephrotoxicity results from apoptosis and necrosis of renal
tubular epithelial cells, and direct damage to the glomerulus as
well. The mechanisms involved are complex.65 Recovery can
occur when the aminoglycoside is removed, but because ami-
noglycosides accumulate in renal tissue, the insult can persist
for some time even after treatment is discontinued. Efforts to
understand the mechanism of nephrotoxicity should lead to
improved preventative measures and increased applications for
aminoglycoside antibiotics. Aminoglycosides bind to megalin-
cubulin complexes on the surface of the cell and are endocy-
tosed into the cytoplasm, where they accumulate in lysosomes.
The lysosomes break down, with resultant drug leakage into
the cytoplasm, where interference with mitochondrial function
occurs. Aminoglycosides also bind membrane phospholipids
and alter their turnover. Damaged renal tubular epithelial cells
are shed into and obstruct the tubular lumen, which leads to a
build-up of pressure within the glomerulus, leading to decreased
glomerular filtration rate. Disrupted proximal tubular cell func-
tion leads to delivery of excessive water and electrolytes to
the distal nephron, which in turn triggers tubuloglomerular
feedback, with constriction of afferent arterioles and further
decreases in the glomerular filtration rate.65 Tubuloglomerular
feedback is designed to protect the body from massive loss of
water and electrolytes.
Risk factors for nephrotoxicity include older age, reduced
renal function, concomitant liver disease, dehydration, sodium
depletion, hypokalemia, and concurrently administered neph-
rotoxic drugs, most commonly nonsteroidal antiinflammatory
drugs but also furosemide and cyclosporin. Although genta-
micin nephrotoxicity can occur in young animals,66 young age
is not a contraindication to use of aminoglycosides, which are
used widely to treat human neonatal sepsis.67 The chance of
toxicity increases with more prolonged treatment periods (≥3
days). Gentamicin and amikacin are more toxic than tobramy-
cin. Volume and salt loading do not decrease toxicity, but high
levels of dietary calcium and protein have protective effects.68
The best preventative measure is to avoid these drugs in any

TABLE 8-12
Suggested Chloramphenicol Dosages for Small Animals
Dose Interval
Drug (mg/kg) (hours) Species
Chloramphenicol, 40-50 6-8 D PO, IV, IM
chloramphenicol 12.5-20 12 C Monitor CBC with long-
sodium succinate
Florfenicol 20 6 D, C
22 8 dogs and cats. Doses and
C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs.
patients with preexisting renal disease. If renal disease develops
after initiating treatment, the drug should be substituted with a
safer drug.
Ototoxicity results from damage to the cochlear or the ves-
tibular apparatus and is more likely to occur in animals with
renal impairment or that are dehydrated. Cochlear toxicity
results from damage to the hair cells of the organ of Corti,
whereas vestibular toxicity results from damage to hair cells at
the tip of the ampullae cristae. Auditory toxicity may be more
common in dogs, whereas cats tend to develop vestibular toxic-
ity.69 Cochlear toxicity in dogs and cats may be underrecog-
nized, because it may be overlooked by pet owners. In human
patients, signs of vestibular toxicity can disappear with time
because of compensatory mechanisms. Ototoxicity is generally
irreversible, but in some human patients, gradual improvement
in hearing can occur.70
Neuromuscular blockade is a rare but potentially life-­
threatening complication of aminoglycoside treatment. Severe
neuromuscular blockade has not been reported in cats and dogs,
but experimental studies show it does occur when aminoglyco-
sides are used in conjunction with some anesthetic agents.71-73
Neuromuscular blockade results from decreased release of
acetylcholine at neuromuscular junctions, and decreased sensi-
tivity of the postsynaptic neuron to acetylcholine. Autonomic
blockade can also occur. The use of aminoglycosides should be
avoided in animals with neuromuscular disease such as myas-
thenia gravis, because aminoglycosides have the potential to
exacerbate the clinical signs.74
Measurement of serum aminoglycoside concentrations can
be used to ensure adequate plasma concentrations or to mini-
mize toxicity.75 Assays are offered by specialized veterinary
pharmacology laboratories or through human hospitals. Ami-
noglycoside doses that result in peak plasma concentrations
approximately 8 to 10 times the MIC produce the optimal bac-
tericidal effect. The effective volume of distribution for amino-
glycosides is often higher in septic patients or animals that have
a “third space” problem (such as pleural effusion), which may
necessitate a higher dose. To perform therapeutic drug monitor-
ing, the optimum time points are 1 hour after injection (Cmax),
CHAPTER 8  Antibacterial Drugs 79
CLSI Breakpoints
Route Comments (µg/mL)
Avoid long-term use in cats. ≤4 for streptococci,
≤8 for other sus-
term use. Warn owners ceptible bacteria
that human exposure to
chloramphenicol may cause
bone marrow disease. Drug
interactions may occur (see
text). Avoid in animals
with hepatic failure.
PO, IM Not approved for use in See chlorampheni-
col.
adverse effects have not
been well studied.
followed by two additional time points in order to estimate the
slope of the elimination curve. Trough levels could also be per-
formed to minimize the chance of toxicity, with the recommen-
dation that if high trough levels are detected, the drug should
be discontinued. In patients with normal renal function, trough
levels with once-daily dosing should be in the “undetectable”
range.
Chloramphenicol
Mechanism of Action and Spectrum of Activity
Chloramphenicol binds to the 50S subunit of the bacterial
ribosome and inhibits bacterial protein synthesis. It is usually
described as bacteriostatic, but there is some evidence that
chloramphenicol may be more bactericidal than previously
thought. Like other ribosomal inhibitors, chloramphenicol
has a broad spectrum of activity, which includes gram-positive
and gram-negative bacteria, anaerobes, and some rickettsial
pathogens. Because chloramphenicol can cause aplastic anemia
in humans, its use in humans has greatly diminished, and it is
only used for the treatment of MDR bacterial infections where
few or no other antimicrobial drugs are useful. Thiamphenicol
and florfenicol are related compounds, the availability of which
varies from country to country. Although thiamphenicol was
initially promoted as not inducing aplastic anemia in humans,
cases of aplastic anemia have now been identified.76 Florfenicol
is more active than chloramphenicol and thiamphenicol, but has
a shorter half-life in dogs and thus requires more frequent dos-
ing (e.g., more often than every 8 hours) in this species. It was
developed for food animal species where use of chlorampheni-
col is illegal.
Resistance to chloramphenicols results from porin mutations,
drug efflux, or production of chloramphenicol acetyltransferase
enzymes, which inactivate the antibiotic.77 Resistance to one
chloramphenicol derivative predicts resistance to the others.
Clinical Use
Doses are shown in Table 8-12. Chloramphenicol is absorbed
orally with or without food. Tablets and capsules have simi-
lar oral absorption, but the extent of oral absorption varies
80 SECTION 2  Antiinfective Therapy
considerably among dogs. Chloramphenicol distributes well to
extracellular fluids of most tissues. Because of its high degree
of lipid solubility, it also diffuses to tissues with barriers, such
as the CNS and the eye. Topical ophthalmic preparations
achieve high concentration in the aqueous humor. In dogs, most
absorbed chloramphenicol is metabolized by glucuronidation in
the liver, with inactive metabolites excreted by the kidney. The
use of chloramphenicol should therefore be avoided in animals
with hepatic failure. Provided renal failure is not present, suf-
ficient active drug is excreted in the urine to enable treatment
of UTIs that are resistant to other drugs, such as methicillin-
resistant staphylococci and resistant enterococci.
Adverse Effects
Exposure of humans to even small amounts of chloramphenicol
has the potential to lead to irreversible aplastic anemia, an idio-
syncratic reaction, weeks to months after discontinuing treat-
ment. The reaction is rare (1 in 24,000 to 1 in 40,000 exposed
humans) and most commonly occurs when human patients are
treated with oral formulations.78 Whether use of ophthalmic
preparations in human patients poses a risk has been contro-
versial.79 Nevertheless, it is recommended that owners of pets
that are being treated with chloramphenicol wear gloves when
handling the drug and to avoid splitting tablets at home. Chlor-
amphenicol commonly causes gastrointestinal adverse effects in
dogs and cats, including anorexia, hypersalivation, and vomit-
ing. High doses or prolonged treatment can result in reversible
bone marrow suppression, which usually resolves within days
after discontinuation of treatment. High doses and prolonged
treatment are needed to produce this effect in dogs, but cats are
more susceptible and can be at risk within 2 weeks of adminis-
tration. Chloramphenicol is a potent inhibitor of P450 enzymes,
and so the potential for significant drug interactions exists. Flor­
fenicol has not been widely used in dogs and cats, and adverse
effects have not yet been reported.
Macrolides and Lincosamides
Classification and Mechanism of Action
The macrolides and lincosamides (clindamycin and lincomycin)
are not chemically related but possess similar mechanisms of
action, resistance, and antimicrobial activity. Macrolides and lin-
cosamides inhibit protein synthesis by binding to the 50S subunit
of bacterial ribosomes. Because they are weak bases, they concen-
trate in the relatively acidic interior of leukocytes. The high con-
centrations in leukocytes may also produce immunomodulatory
effects. Macrolides, particularly the newer agents such as azithro-
mycin, can impair leukocyte function, interfere with superoxide
production by mast cells, suppress proinflammatory cytokine
release, and produce apoptosis.80,81 These effects may suppress
inflammatory reactions in some tissues, but the significance of
these effects has not been explored in small animal infections.
The first macrolide antibiotic to be discovered was eryth-
romycin, a derivative of Streptomyces erythreus. Despite the
common occurrence of gastrointestinal adverse effects, poor
gastrointestinal absorption, and an extremely short half-life,
erythromycin continues to have application in human medicine
because of its safety in pregnancy and utility in human patients
who are allergic to penicillin. Because these concerns are less
important in dogs and cats, erythromycin is less commonly used
in small animal patients.
Since the discovery of erythromycin, several semisynthetic
macrolides have become available. Azithromycin and other new
macrolide antibiotics, such as clarithromycin and roxithromy-
cin, have been referred to as “advanced-generation” macrolides.
These have an extended spectrum of activity, with greater effi-
cacy against atypical and some gram-negative bacteria, a longer
duration of antimicrobial activity, … improved gastrointestinal
absorption, greater intracellular concentrations, and a marked
reduction in gastrointestinal adverse effects. Clarithromycin has
improved activity against streptococci and staphylococci com-
pared with erythromycin.
Azithromycin is an azalide, because unlike other macrolides,
it contains nitrogen. Azithromycin is extensively concentrated
within cells and is retained in tissues for prolonged periods, with
a half-life of 30 hours in the dog and 35 hours in the cat. In
human patients, a 5-day course provides therapeutic tissue con-
centrations for at least 10 days. Although azithromycin has an
improved spectrum against gram-negative bacteria, it has less
activity against gram-positive bacteria than erythromycin.82,83
Susceptibility to erythromycin often predicts susceptibility
to other macrolide antimicrobial drugs. Resistance to macro-
lides and lincosamides results from decreased bacterial per-
meability (for gram-negative bacteria), alteration in the target
site, increased drug efflux, and enzymatic inactivation of cer-
tain macrolides by bacterial esterases. Alteration in the target
site involves production of a ribosomal methylase that adds a
methyl group to the 50S subunit RNA, preventing the macro-
lide from binding to the ribosome. Expression of the methylase
enzyme confers high-level resistance to azithromycin, clarithro-
mycin, and also clindamycin.
Clinical Use
Macrolides and lincosamides are primarily used to treat gram-
positive bacterial infections. They are generally considered to
be bacteriostatic. Doses are shown in Table 8-13. Erythromycin
tablets have a protective coating to prevent degradation by gas-
tric acid. After absorption, erythromycin is distributed well to
most tissues and body fluids, with the exception of the CNS and
urine. It is excreted in high concentrations in bile, followed by
enterohepatic circulation, and ultimately eliminated in the feces.
Non-absorbed drug is also present in the feces. It has activity
against some anaerobes, spiral bacteria (particularly Campylo-
bacter), and many intracellular bacteria. Tylosin has been used
to treat chronic colitis in dogs84 and is also active against Cam-
pylobacter and enteric Clostridium spp.
Clarithromycin and azithromycin have considerably longer
half-lives than erythromycin. The intracellular concentration of
clarithromycin is about two- to sixfold that in plasma. Clar-
ithromycin is preferred over erythromycin or azithromycin for
treatment of rapidly growing mycobacterial infections in dogs
and cats (usually in combination with rifampin) (see Chapter
44). In humans, clarithromycin is extensively metabolized by
the liver to a variety of specific active and inactive metabo-
lites, with some excretion of active drug and its metabolites
into urine. Formation and excretion of these metabolites have
not been well studied in dogs and cats. Azithromycin is largely
excreted unchanged in bile.
Because of its prolonged tissue retention, serum concentra-
tions of azithromycin do not reflect tissue concentrations, and
so the antibiotic can still be effective even if the serum con-
centration is less than the MIC. Intracellular concentrations
of azithromycin are 10- to 100-fold those in serum, concen-
trations that are considered bactericidal. Concentrations can
remain high within tissues several days after the drug has been
 CHAPTER 8  Antibacterial Drugs 81

TABLE 8-13
Suggested Macrolide and Lincosamide Dosages for Small Animals
Dose Interval CLSI Breakpoints
Drug (mg/kg) (hours) Species Route Comments (µg/mL)
Erythromycin 10-20 8 D, C PO Do not administer to rabbits ≤0.25 for streptococci and
or rodents, as this may cause ≤0.5 for other suscep-
fatal diarrhea. Drug interac- tible organisms
tions may occur (see text).
Tylosin 7-15 12-24 D, C PO See erythromycin. For colitis in
8-11 12 IM dogs, use 20 mg/kg q8h with
food, and taper to q24h if a
response occurs.*
Clarithromycin 7.5 12 D, C PO Drug interactions may occur ≤2 for staphylococci.
(see text). Consider dosage Respiratory cures may
reduction in severe renal be possible for organ-
failure. isms with MICs up to 8,
because drug concen-
trates within the lung.
Azithromycin 5-10 24 D, C PO ≤ 2 for susceptible bacte-
ria. Serum MIC values
may not predict tissue
concentrations.
Lincomycin 15-25 12 D, C PO See erythromycin. A dose of 0.25 for streptococci and
­hydrochloride 10 mg/kg PO q12h has been ≤ 0.5 for other suscep-
suggested for pyoderma. tible organisms
Clindamycin 11-33 12 D PO See erythromycin. For IV See lincomycin.
­hydrochloride, 11 24 C PO administration, dilute 1:10 in
clindamycin 10 12 D, C IV, IM 0.9% saline and administer
­phosphate over 30-60 minutes. The oral
suspension may be unpalat-
able to cats. For toxoplasmo-
sis, use 25 mg/kg q12h PO.

C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; MIC, minimum inhibitory concentration.
*20 mg/kg is equal to 1/8th teaspoon of tylosin phosphate (Tylan) for a 20-kg dog.

discontinued. Despite the improved spectrum of activity of
clarithromycin and azithromycin, with activity against some
gram-negative bacteria and intracellular pathogens, many of
these organisms are resistant. The primary indication for these
antibiotics in small animals is in combination with other drugs
such as rifampin for treatment of mycobacteriosis or bartonel-
losis, or atovaquone for treatment of certain systemic protozoal
infections such as babesiosis and cytauxzoonosis. Azithromycin
has been used to treat secondary bacterial infections in cats with
upper respiratory tract disease because its long half-life permits
convenient dosing on an every-48- or every-72-hour basis.
Despite its popularity for treating upper respiratory infections
in cats, one study showed no benefit to this protocol over use of
amoxicillin,85 and the potential for overuse of azithromycin in
this setting raises concerns in regard to selection for resistance
to azithromycin.
Lincomycin is active against gram-positive bacteria and has
been used to treat staphylococcal pyoderma. However, the
use of lincomycin has largely been replaced by clindamycin,
which has similar activity, favorable pharmacokinetics, and
widely available formulations (tablets, capsules, oral liquid, and
injection). Clindamycin is also used to treat anaerobic bacterial
infections, as well as toxoplasmosis and neosporosis (see Chap-
ters 72 and 73).
Adverse Effects
Although otherwise very safe, erythromycin frequently causes
vomiting, decreased appetite and nausea because of stimu-
lation of receptors of the gastrointestinal hormone motilin,
which increases gastrointestinal smooth muscle activity.86
Like other poorly absorbed oral antimicrobials, it can pro-
duce diarrhea due to changes in the gastrointestinal flora.
Although diarrhea and pseudomembranous colitis are seri-
ous complications of clindamycin treatment in people, these
problems are uncommon in small animals. Adverse effects are
rarely reported for clarithromycin and azithromycin. Eryth-
romycin and azithromycin inhibit P450 enzymes and so can
increase the concentration of other drugs that are adminis-
tered concurrently.
Clindamycin and lincomycin can cause vomiting and diar-
rhea, especially when clindamycin is used at high doses. Oral
clindamycin hydrochloride can cause esophagitis in cats.87,88
82 SECTION 2  Antiinfective Therapy
TABLE 8-14
Suggested Linezolid Dose for Small Animals
Drug Dose (mg/kg) Interval (hours) Species
Linezolid* 10 8 D
CLSI, Clinical and Laboratory Standards Institute; D, dogs.
*The use of linezolid should be reserved for gram-positive infections that are susceptible to linezolid but resistant to all other reasonable alterna-
tives, on the basis of culture and susceptibility testing.
Oxazolidinones (Linezolid)
Mechanism of Action and Spectrum of Activity
Linezolid is a synthetic antibiotic used to treat life-threatening
infections caused by gram-positive bacteria that are resistant to
other antimicrobial drugs, especially methicillin-resistant and
vancomycin-resistant staphylococci. Linezolid binds to the 50S
ribosome and prevents formation of the initiation complex for
protein synthesis.89 This is a unique mechanism, because other
protein synthesis inhibitors interfere with polypeptide exten-
sion. Resistance to other protein synthesis inhibitors does not
correlate with resistance to linezolid. Rarely, resistance to line-
zolid is due to modification of the drug’s target site, but this
is extremely rare because several step mutations are necessary
before resistance can occur. The high cost of oral linezolid cur-
rently limits its application in small animals. Currently, its use
has been limited to dogs and cats with resistant staphylococ-
cal or enterococcal infections for which other antibiotics are
not effective or have produced adverse effects. Linezolid has
favorable pharmacokinetics in dogs,90 with nearly 100% oral
absorption, which is not influenced by food (Table 8-14). The
pharmacokinetics in cats has not been published. Because line-
zolid is bacteriostatic, plasma concentrations should be main-
tained above the MIC throughout the dosing interval.
In human patients, adverse effects include vomiting, diar-
rhea, and, rarely, thrombocytopenia or reversible pure red
cell aplasia (PRCA). Linezolid is a type A monoamine oxidase
inhibitor and so can interact with serotonin reuptake inhibi-
tors.92 Linezolid appears to be well tolerated by dogs and cats.
Despite the concern about monoamine oxidase inhibition and
bone marrow suppression in human patients, these effects have
not yet been documented in dogs or cats, although one of the
authors is aware of a cat that developed reversible PRCA after
treatment with linezolid.
Tetracyclines
Mechanism of Action
Tetracyclines are bacteriostatic, time-dependent antibiotics that
have a broad spectrum of activity that includes gram-positive
and gram-negative bacteria, some anaerobes, and atypical and
intracellular pathogens such as spirochetes, Mycoplasma spp.,
and rickettsiae. In contrast to the macrolides and clindamycin,
which bind to the 50S ribosomal subunit, tetracyclines inhibit
bacterial protein synthesis by binding to the 30S subunit. Thus,
resistance to macrolides does not imply resistance to tetracy-
clines. Examples of tetracyclines used to treat dogs and cats are
CLSI Breakpoints
Route Comments (µg/mL)
PO, IV If administered with other ≤4 for staphylococ-
drugs IV, the adminis- ci, ≤2 for Entero-
tration line should be coccus spp.
flushed first.
tetracycline, oxytetracycline, and the semisynthetic tetracycline
doxycycline. Doxycycline and minocycline are longer-acting tet-
racyclines that are more lipophilic than the older tetracyclines.
Doxycycline and minocycline also have antiinflammatory and
immunomodulatory properties that result from inhibition of
inducible nitric oxide synthase and proinflammatory cytokines
such as TNF-α.91 These properties have led to the use of tet-
racyclines for treatment of immune-mediated dermatopathies
in dogs92 and plasmacytic pododermatitis in cats.93 Delayed-
release formulations that produce subantimicrobial doses have
been developed for use in human patients with the inflamma-
tory skin disease rosacea. Although development of tetracycline
resistance in bacterial flora has not been documented in associa-
tion with the use of these preparations, the possibility has not
been ruled out.94
Resistance to tetracyclines primarily results from porin
mutations that exclude tetracyclines from the bacterial cell, and
increased drug efflux, mediated by the tetK gene. Some bacte-
ria with tetK may still be susceptible to minocycline. Resistance
mediated by tetM confers resistance to all tetracyclines.95 In
diagnostic laboratories, tetracycline is used to assess the sus-
ceptibility of bacteria to all other tetracyclines except the gly-
cylcycline antibiotic tigecycline, a new antibiotic derived from
minocycline that is reserved for treatment of multidrug-resistant
bacterial infections.96 In addition, resistance to doxycycline
does not always imply resistance to minocycline.97
Clinical Use
Doses are shown in Table 8-15. Doxycycline is the most com-
monly used tetracycline in dogs and cats and has fewer adverse
effects when compared with other tetracyclines. It is the drug
of choice for treatment of a variety of tick-borne infectious dis-
eases, feline chlamydiosis, salmon poisoning disease, and lep-
tospirosis. The long half-life of doxycycline permits once-daily
dosing, although twice-daily dosing is frequently recommended.
Doxycycline is well absorbed from the gastrointestinal tract and
distributed to a variety of tissues, including the lung, bronchial
secretions, liver, kidney, and, to some extent, the CNS. If doxy-
cycline is not available, minocycline can be used as a substitute.
Minocycline is more lipophilic than doxycycline, it is better
absorbed orally, and can be administered at similar doses as
doxycycline. Absorption of other tetracyclines from the gastroin-
testinal tract is not as efficient. All tetracyclines are concentrated
in bile. Sufficient drug is excreted in the urine to permit treatment
of UTIs caused by bacteria that are resistant to other drugs.
 CHAPTER 8  Antibacterial Drugs 83

TABLE 8-15
Suggested Tetracycline Dosages for Small Animals
Dose Interval CLSI Breakpoints
Drug (mg/kg) (hours) Species Route Comments (µg/mL)
Tetracycline 15-20 8 D, C PO Do not mix with food containing ≤2 for streptococci, ≤4 for other
4.4-11 IV, IM cations such as calcium, zinc, susceptible bacteria. Consider
magnesium, iron, aluminum. use of ≤1 for susceptible bacteria.
Oxytetracycline 20 12 D, C PO See tetracycline. See doxycycline.
7.5-10 IV
Doxycycline 5 12 D, C PO, IV For IV administration, dilute in ≤2 for streptococci, ≤0.25 for
10 24 100 to 1000 mL of LRS or 5% Staphylococcus pseudinterme-
dextrose and administer over dius, ≤4 for other susceptible
1 to 2 hours. bacteria
Minocycline 5-12.5 12 D, C PO See doxycycline, but the pharma-
cokinetics of minocycline in dogs
and cats require further study.

C, Cats; CLSI, Clinical and Laboratory Standards Institute; D, dogs; LRS, lactated Ringer solution.

A should be used. Compounded, high-concentration (e.g., 33.3
B Intravenous preparations of doxycycline are available but
FIGURE 8-4  A, Esophageal stricture in a 9-year-old female spayed domestic shorthair
that had been treated with doxycycline for possible hemoplasmosis. Regurgitation after
eating commenced shortly after treatment was initiated. B, The stricture was treated via
repeated balloon dilatation; the image is immediately after the ballooning procedure was
performed.
Adverse Effects
The most common adverse effects of tetracyclines in dogs and
cats are vomiting, decreased appetite, nausea, and diarrhea.
These may be reduced when doxycycline hyclate tablets are
administered intact (rather than being split or crushed) and by
administering doxycycline with food. Administration of other
tetracyclines with food can lead to significantly reduced drug
absorption, because of extensive binding with divalent and
trivalent cations in the gastrointestinal tract. The hyclate salt
of doxycycline (doxycycline hydrochloride) has been associ-
ated with esophagitis and esophageal strictures in cats (Figure
8-4),98 and life-threatening esophageal ulceration in a dog.99
This is also a complication in people.100 A bolus of water or
food should be administered immediately after dosing in order
to prevent this complication, or a suspension rather than tablets
mg/mL or 167 mg/mL) suspensions in aqueous vehicles have
limited stability; suspensions in some vehicles do not retain their
potency beyond 7 days.101
In dogs and cats with renal failure, tetracyclines other than
doxycycline should be avoided. Doxycycline is excreted in the
intestinal tract in this situation. Tetracyclines other than dox-
ycycline can interfere with bone growth and cause significant
and permanent gray-brown or yellow discoloration of the teeth
­(Figure 8-5), and so their use is not recommended in puppies
and kittens. Doxycycline is not likely to produce this effect
because it binds calcium less avidly. As a result, the use of dox-
ycycline in human pediatric medicine is now accepted.102 All
tetracyclines, including doxycycline, have the potential to cause
hepatic failure and renal tubular necrosis. Cutaneous reactions
occur rarely in animals treated with tetracycline, but have been
reported in humans.
can be considerably more expensive than the oral formulations.
They must be given over 1 to 2 hours. Thrombophlebitis can
occur at the site of injection, and anaphylactic shock has been
reported following parenteral administration of tetracycline to
a dog.103
84 SECTION 2  Antiinfective Therapy
FIGURE 8-5  Yellow discoloration of the teeth in a “puppy mill” puppy that was pre-
sumably treated with tetracycline antibiotics.
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in the U.S. Military service. Mil Med. 2000;165:316-319.
101. Papich MG, Davidson G. Doxycycline potency after storage in a
compounded formulation for animals. American College of Vet-
erinary Internal Medicine Forum. 2011. Denver, CO, USA.
102. Volovitz B, Shkap R, Amir J, et  al. Absence of tooth staining
with doxycycline treatment in young children. Clin Pediatr (Phila).
2007;46:121-126.
103. Ward GS, Guiry CC, Alexander LL. Tetracycline-induced anaphy-
lactic shock in a dog. J Am Vet Med Assoc. 1982;180:770-771.
CHAPTER 9
Antifungal Drugs
Jane E. Sykes and Mark G. Papich
w KEY POINTS
• C  ompared to antibacterial drugs, the range of antifungal drug
classes available is very limited, with most systemic treatment
accomplished with the azole group of medications. The azoles
have a similar mechanism of action and share other properties
such as spectrum of activity, pharmacokinetics, tissue penetra-
tion, and adverse effect profiles. Proper understanding of these
factors is most likely to result in successful treatment with mini-
mal adverse effects.
• Drug interactions occur with many antifungal drugs.
• Although therapeutic drug monitoring would improve treat-
ment with the azole antifungals and 5-flucytosine, this is rarely
INTRODUCTION
The use of antifungal drugs in human and veterinary medicine
has increased in recent years. In humans, increased suscepti-
bility to fungal infections has arisen because of immunosup-
pression secondary to HIV infection or treatment with potent
immunosuppressive drugs. In animals, opportunistic fungal
infections also result from immunosuppression, and the impor-
tance of fungal infections in dogs and cats is increasingly recog-
nized. Antifungal drugs used in dogs and cats are generally not
approved for use in these species. Therefore, veterinarians use
human antifungal drugs off-label, with indications and dosing
protocols often extrapolated from the human use. Because of
differences in pharmacokinetics among species for these drugs,
and differences in susceptibility to adverse effects in dogs and
cats compared to people, this approach is not optimal. Fortu-
nately, the clinical experience and pharmacokinetic data have
expanded for these human drugs, which will improve anti-
fungal treatment in animals. Efforts also have focused on the
development of new, less toxic, and more efficacious antifungal
drugs. This chapter reviews the classification, mechanisms of
action, spectrum of activity, resistance mechanisms, tissue pen-
etration, clinical use, and adverse effects of the major systemic
antifungal drugs used to treat dogs and cats. Topical antifungal
treatments are reviewed in the relevant chapters of this book.
Basic principles of antimicrobial drug treatment are discussed
in Chapter 6.
Antifungal drug treatment is often more prolonged than
antibacterial drug treatment. One reason for this difference is
that fungal organisms grow more slowly, and the drugs used
(primarily azoles) are fungistatic, not fungicidal. Therefore, long-
term treatment is needed to inhibit fungal growth and allow for
the animal’s immune system, which is often compromised, to
eradicate the infection. Treatment of animals with deep mycoses
done because of variable availability of clinical assays and
uncertainty in interpretation of the measured concentrations.
• Fungal infections often require prolonged treatment durations.
This may be costly for some pet owners and can increase the
risk of adverse effects to pets.
• Fungal organisms that are resistant to certain antifungal drugs
are increasingly recognized. Although antifungal suscepti-
bility testing can identify resistant organisms, this is often
not performed because there are no standards for veterinary
pathogens and drugs used in veterinary medicine, and few
laboratories perform these tests.
with antifungal drugs must often be continued for months, and
in some animals it may be weeks before clinical improvement is
evident. Sometimes, disease worsens in the first week of treat-
ment, because of the host’s inflammatory response to killed
organisms. This may have severe consequences for the host
when there is extensive involvement of the pulmonary paren-
chyma or the central nervous system (CNS). Concurrent treat-
ment with a nonsteroidal antiinflammatory drug (NSAIDs) or,
when CNS involvement is present, judicious use of a short course
of antiinflammatory glucocorticoids may improve outcome in
these situations.1
The prognosis for animals with disseminated mold infec-
tions that have reversible underlying immunosuppression (such
as drug-induced immunosuppression) may be better than the
prognosis for animals with no apparent underlying immuno-
suppressive disease. This is because the latter group of animals
are likely to have underlying (irreversible) genetic defects in
immunity.
Azole Antifungals
Mechanism of Action, Classification,
and Spectrum of Activity
Azole antifungal drugs inhibit sterol 14α-demethylase, a cyto-
chrome P450–dependent fungal enzyme involved in synthesis of
ergosterol, a key component of the fungal cell wall, from lanos-
terol. The result is the accumulation of 14α-methylsterols, which
disrupt the fungal cell membrane (Figure 9-1). The majority of
the adverse effects and drug interactions observed with these
drugs relate to the cross-inhibition of mammalian P450 enzymes.
All of the azole antifungals have the potential to be teratogenic,
and their use should be avoided in pregnancy.
Azole antifungals are classified as imidazoles or triazoles
based on whether they possess two or three nitrogen molecules
87
88 SECTION 2  Antiinfective Therapy
FIGURE 9-1  Mechanism of action of antifungal drugs. 5FC, 5-flucytosine; 5FUTP, 5-fluorouridine triphosphate; 5FdUMP, 5-fluorodeoxyuridine monophosphate; A, amphotericin B;
E, ergosterol. Modified from Rex JH, Stevens DA. Systemic antifungal agents. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas and Bennett’s Principles and Practice of Infectious
Diseases, 7 ed. Philadelphia, PA: Churchill Livingstone Elsevier; 2011:549-563.
in their azole ring. Ketoconazole, enilconazole, and clotrimazole
are imidazoles. The latter two drugs have poor oral bioavailabil-
ity and are used topically in veterinary medicine for the treat-
ment of superficial mycoses (see Chapters 65 and 67). Triazole
antifungal drugs, such as itraconazole and fluconazole, are more
slowly metabolized and have less impact on mammalian sterol
synthesis than do imidazoles. Imidazoles and triazoles have
been widely used to treat a variety of mycoses, which include
candidiasis, cryptococcosis, blastomycosis, histoplasmosis, coc-
cidioidomycosis, dermatophytosis, sporotrichosis, and aspergil-
losis (Tables 9-1 and 9-2).
Unfortunately, resistance to azole antifungal drugs has
emerged among some fungi. Resistance results from mutations
in the gene encoding the demethylase enzyme, increased produc-
tion of C-14α demethylase, and increased azole efflux by fungal
cell membrane transporters. Methods for in  vitro susceptibil-
ity testing have become more standardized,2,3 with improved
correlation between the results of in  vitro susceptibility test-
ing and clinical response, but breakpoints (criteria that define
susceptible versus resistant minimum inhibitory concentration
values) are still needed for many drug-fungus combinations.
Increasingly, it is apparent that resistance to one azole does not
always imply resistance to other azole antifungal drugs.
Ketoconazole
Spectrum of Activity and Clinical Use
The use of ketoconazole has largely been replaced by itracon-
azole for treatment of many mycoses, because of the greater
toxicity and reduced efficacy of ketoconazole when compared
with triazole antifungal drugs. Because of its low cost, keto-
conazole continues to be used in veterinary medicine when the
cost of other antifungal drugs is prohibitive to the client, and it
remains efficacious for treatment of Malassezia dermatitis4 and
feline nasal and cutaneous cryptococcosis. The absorption of
ketoconazole is improved when it is administered with food,
but it is inhibited by concurrent use of antacids. Ketoconazole
is highly protein bound and is metabolized extensively by the
liver. Nevertheless, moderate hepatic dysfunction does not alter
blood levels of ketoconazole. Inactive products are excreted in
bile and, to a lesser extent, the urine. Because of poor CNS pen-
etration, it is ineffective for treatment of meningeal cryptococ-
cosis and aspergillosis.5
 CHAPTER 9  Antifungal Drugs 89

TABLE 9-1
Spectrum of Activity and Tissue Distribution of Different Antifungal Drugs
Drug Spectrum of Activity Tissue Distribution
Ketoconazole Dimorphic fungi, Malassezia. Ineffective for aspergillosis Skin, bone, joint, lung. Poor CNS penetration
Itraconazole Dimorphic fungi and molds, Malassezia spp. Skin, bone, lung. May enter the CNS and
eye with inflammation
Fluconazole Some Candida spp., Malassezia spp., some dimorphic fungi. Widely distributed, including to the skin,
Poor activity against molds. Aspergillus spp. are intrinsi- lung, CNS, urine, and eye
cally resistant
Voriconazole Dimorphic fungi, yeasts, and molds with the exception of CNS, eye, lung, bone
Sporothrix schenckii and zygomycetes
Posaconazole Dimorphic fungi, yeasts, and molds including zygomycetes Widely distributed
Amphotericin B Broad spectrum. Also active against Leishmania Limited penetration of the CNS and eye
5-Flucytosine Cryptococcus and Candida spp. Widely distributed, which includes the CNS,
eye, and urine
Griseofulvin Dermatophytes Concentrates in skin
Terbinafine Activity highest for dermatophytes. To a lesser extent may Concentrates in skin and hair
have activity against other dimorphic and filamentous fungi
Caspofungin Candida and Aspergillus spp. Not active against Cryptococcus Widely distributed. Poor penetration of the
spp. or when given alone to treat Coccidioides CNS and eye

CNS, Central nervous system.

TABLE 9-2
Suggested Azole Antifungal Drug Doses for Dogs and Cats
Drug Dose Interval Species Route Comments
(mg/kg) (hours)
Ketoconazole 10 to 15 12 D PO Do not administer to pregnant animals. Monitor liver enzymes monthly
5 to 10 C during treatment. Drug interactions may occur (see text). Inhibits
adrenal function. Antacids impair absorption.
Itraconazole 5 12 to 24 D, C PO Do not administer to pregnant animals. Monitor liver enzymes monthly
during treatment. Drug interactions may occur. Use of the oral suspen-
sion warrants dose reduction to 3 mg/kg. Monitor serum drug concen-
trations after 2 weeks if there is inadequate response to treatment (see
text). Compounded formulations are unstable.
Fluconazole 5 to 10 12 D PO Do not administer to pregnant animals. Monitor liver enzymes monthly
50 mg/cat 12 to 24 C during treatment. Drug interactions may occur.
Voriconazole 4 to 5 12 D PO Do not use in cats. Use cautiously in animals with liver disease. Also see
fluconazole. Consider therapeutic drug monitoring (see text).
Posaconazole 5 to 10 12 to 24 D PO Absorption may be improved when daily dose is split into 2 to 4 doses.
5 24 C Consider therapeutic drug monitoring. Also see fluconazole. Antacids
impair absorption.

C, Cats; D, dogs.

Adverse Effects vomiting, lethargy, increasing activities of serum ALT and

The most common adverse effects of ketoconazole in dogs and
cats are vomiting, anorexia, lethargy, and diarrhea.6,7 Admin-
istration of ketoconazole with food may reduce gastrointes-
tinal adverse effects. Mild increases in the activity of serum
transaminases occur commonly during treatment, but do not
warrant discontinuation of the drug. Less commonly, ketocon-
azole causes hepatitis, which may be accompanied by anorexia,
ALP, and hyperbilirubinemia. The drug should be discontin-
ued if this occurs and serum chemistry values checked 1 to 2
days later. Hepatitis can occur at any time, and the onset may
be extremely rapid. Pruritis and cutaneous erythema have been
reported in fewer than 1% of dogs treated with ketoconazole.6
Lightening of the hair coat color and cataract formation occur
rarely.
90 SECTION 2  Antiinfective Therapy
Ketoconazole is a potent inhibitor of mammalian cytochrome
P450 enzymes and efflux transporter proteins such as P-glyco-
protein. It also inhibits testosterone and cortisol synthesis. As a
result, it has been used to treat pituitary-dependent hyperadre-
nocorticism in dogs8 and also to deliberately inhibit the metabo-
lism of cyclosporin, allowing a reduction in dose and, therefore,
cost,9 although there are concerns about whether use of keto-
conazole in this way could contribute antifungal drug resistance
and it increases the risk of adverse drug reactions. Transient
infertility can occur during treatment of intact male animals.
Ketoconazole can interfere with P-glycoprotein transport of
ivermectin, which predisposes dogs to ivermectin toxicity.6,10
Itraconazole
Spectrum of Activity and Clinical Use
Itraconazole is one of the most widely used azoles in veteri-
nary medicine. It has been used to treat blastomycosis, sporo-
trichosis, aspergillosis, coccidioidomycosis, dermatophytosis,
histoplasmosis, phaeohyphomycosis, paecilomycosis, crypto-
coccosis, and Malassezia infections. It is available in capsules
(which contain itraconazole granules) and an oral solution. The
IV solution has been withdrawn.11 Itraconazole in capsules is
best absorbed when given with food because the acid secretion
stimulated with feeding increases the drug solubility, which is
necessary for dissolution and absorption. However, the oral
solution is complexed with cyclodextrin to improve solubility,
and administration of food does not influence absorption of
this formulation. Absorption of the oral solution is consistently
better than absorption of the capsule formulation, regardless
of feeding.12 As with ketoconazole, concurrent administration
of gastric acid suppressants (H2-blockers, proton pump inhibi-
tors) may reduce absorption of the oral capsule. In cats, the oral
solution is very well absorbed, and dose reduction is indicated
to reduce the chance of toxicity (see Table 9-2). In addition to
hydroxypropyl-β-cyclodextrin, this formulation contains sor-
bitol, propylene glycol, hydrochloric acid, cherry flavor, and
saccharin, which may be unpalatable for cats. In Europe and
Canada an oral solution for cats is available (Itrafungol, 10 mg/
mL). Compounded formulations prepared by pharmacists have
highly variable, poor, and even negligible oral absorption in ani-
mals, in addition to being unstable formulations. As a result,
compounded formulations should never be used.
Like ketoconazole, itraconazole undergoes hepatic metabo-
lism and inhibits metabolism of other P450-dependent drugs
(e.g., cisapride, diazepam, cyclosporin). It is the only triazole
antifungal drug that is converted to an active metabolite,
hydroxylitraconazole.11 Itraconazole is highly (99%) plasma
protein bound and does not appear in urine, cerebrospinal fluid
(CSF), or ocular tissues, although penetration of the CSF and
eye can occur in the presence of inflammation. Itraconazole
accumulates in the skin and claws and is the drug of choice
for treatment of dermatophytosis in cats. Itraconazole is also
a good choice for treatment of fungal osteomyelitis. Although
loading doses have been recommended, these initial high doses
can increase the risk of toxicity and do not appear to improve
outcome.13 Advanced liver disease increases itraconazole con-
centrations. Because of the variable absorption of oral itracon-
azole, monitoring of steady-state plasma concentrations (e.g.,
3 weeks after initiating treatment) may be helpful if clinical
responses are suboptimal.14 Trough plasma itraconazole con-
centrations should be 0.5 to 1 µg/mL as determined using high-
performance liquid chromatography.15,16
FIGURE 9-2  Submandibular ulcerative skin lesions in a golden retriever that had
been treated with itraconazole for systemic blastomycosis.
Adverse Effects
The most common adverse effects of itraconazole in dogs and
cats are vomiting and anorexia. Division of the total dose for
twice-daily administration has been associated with decreased
gastrointestinal signs and improved absorption in human
patients.5 Gastrointestinal adverse effects may be more common
with the oral solution. Mild to moderate increases in serum ALT
activity commonly occur during treatment. Provided these are
not accompanied by inappetence, treatment need not be discon-
tinued. Significant hepatotoxicity, accompanied by anorexia,
vomiting, and hyperbilirubinemia, is less likely to occur than
with ketoconazole but has been reported in humans as well as
cats and dogs.13,17-19 Hepatotoxicity more commonly occurs in
dogs treated with 10 mg/kg/day.13 Ulcerative skin lesions can
occur in dogs, especially when doses of 10 mg/kg/day or higher
are used (Figure 9-2). Development of hepatitis or severe cuta-
neous ulceration should prompt discontinuation of the drug. It
may be possible to reinstitute treatment at a lower dose once the
adverse effects have resolved. Occasionally mild, focal cutane-
ous ulcerative lesions can resolve spontaneously, without dis-
continuation of treatment.
Itraconazole does not suppress adrenal and testicular func-
tion like ketoconazole, but can inhibit the metabolism of
other P450 enzyme-dependent drugs, including cyclosporine,
digoxin, cisapride, and vinca alkaloids. It can also interfere
with ­P-glycoprotein transport of ivermectin, which results in
increased plasma ivermectin concentrations.20
Fluconazole
Spectrum of Activity and Clinical Use
Fluconazole is the least active azole antifungal drug and has the
narrowest spectrum. The activity of fluconazole is limited to
some Candida spp., Cryptococcus spp., Malassezia spp., and
some dimorphic fungi. Fluconazole has poor activity against
molds, and Aspergillus species are intrinsically resistant to flu-
conazole. Some Cryptococcus isolates are resistant to flucon-
azole, and resistance can develop during treatment. Itraconazole
is preferable to fluconazole for treatment of histoplasmosis,
blastomycosis, sporotrichosis, and dermatophytosis, although

fluconazole has been successfully used to treat coccidioidomy-
cosis, blastomycosis, and Malassezia infections in dogs.19
Fluconazole is available as tablets, an oral suspension, and
as an IV solution. The availability of generic fluconazole has
greatly reduced its cost and increased its use in veterinary medi-
cine. Approved generic fluconazole tablets are bioequivalent to
the brand name formulation in people. Similar bioequivalence is
likely in animals. Fluconazole is more water-soluble and stable
than itraconazole, and the compounded oral suspensions can
be used in animals with a 14-day beyond-use-day (BUD). Some
compounded preparations may have reduced activity. Flucon-
azole is almost completely absorbed after oral administration,
and bioavailability is not altered by food or gastric acidity. In
contrast to itraconazole and ketoconazole, fluconazole is only
weakly protein bound. Because it is more water soluble than
other azoles, fluconazole diffuses into body fluids such as saliva,
urine, synovial fluid, and CSF. Therefore, it is the drug of choice
for treatment of susceptible meningeal and urinary tract fungal
infections. Renal excretion accounts for more than 90% of the
elimination of fluconazole. The dosage should be decreased in
animals with renal failure; this is especially important in patients
receiving other P450-dependent drugs.
Adverse Effects
Adverse effects of fluconazole are uncommon to rare. When
they occur, they are similar to those of itraconazole and include
vomiting, anorexia, diarrhea, and increased liver enzymes. Dogs
that fail to tolerate itraconazole may tolerate fluconazole with-
out increases in liver enzymes.19 Cutaneous rash, cytopenias,
and retinal edema with impaired vision have been reported
in human patients.21,22 In contrast to itraconazole, cutaneous
ulceration has not been reported in dogs.
Voriconazole
Spectrum of Activity and Clinical Use
Voriconazole is a second-generation triazole that is derived
from fluconazole. In humans, it is the drug of choice for treat-
ment of invasive aspergillosis.23 It is also used to treat serious
and refractory mold infections caused by Scedosporium spp.,
Paecilomyces spp., and Fusarium spp. Voriconazole is at least
as active as itraconazole against veterinary isolates of Crypto-
coccus spp., Candida spp., and Aspergillus fumigatus.24 It is not
active against Sporothrix spp. or the zygomycetes.
Voriconazole is available as a tablet, an oral suspension, and
an IV solution in a sulfobutylether–β-cyclodextrin base. Like
fluconazole, it has excellent oral bioavailability, but its absorp-
tion is significantly reduced in the presence of food. Voricon-
azole is poorly water soluble and moderately protein bound. It
is extensively metabolized by hepatic cytochrome P450 enzymes
and eliminated into bile. The degree to which active drug enters
the urinary tract in dogs is not clear. It has good CNS pen-
etration. Voriconazole has been used with some success to treat
systemic mold infections in dogs.25 If therapeutic drug monitor-
ing can be performed, the recommended trough concentration
target is between 1 and 5 mg/L, and peak concentrations below
6 mg/L.15,16,23
Adverse Effects
In humans, adverse effects of voriconazole include reversible
visual effects such as photophobia and blurred vision, hallucina-
tions, peripheral neuropathies, and photosensitization, as well
as the same toxicities as other triazoles. Of all the triazoles, it
CHAPTER 9  Antifungal Drugs 91
is the most potent inhibitor of P450 enzymes. Voriconazole can
induce its own metabolism over time.26 Because of its high cost,
adverse effects have not been well documented in dogs. The
author has observed inappetence, increased serum liver enzyme
activities, and, uncommonly, CNS signs, including ataxia and
staring. Tachypnea and marked pyrexia after IV infusion was
observed in one dog. Cats are extremely sensitive to adverse
effects of voriconazole and predictably develop inappetence, as
well as ocular and CNS signs that have included ataxia, pelvic
limb paresis, mydriasis, apparent blindness, decreased pupillary
light responses, and a decreased menace response.27 Cardiac
arrhythmias and hypokalemia also occur in cats. A safe dose
has not been established for cats.
Posaconazole
Posaconazole is an itraconazole analogue that has demonstrated
good efficacy for treatment of several refractory deep mycoses
in animals, which includes aspergillosis and mucormycosis in
cats and invasive aspergillosis in dogs.25,28-30 It has the broadest
spectrum of activity of all the azoles. Its spectrum of activity is
similar to that of voriconazole, but also includes the zygomyce-
tes.11 Posaconazole is available as an oral suspension. As with
itraconazole, its absorption is promoted by the concurrent pres-
ence of fatty food. Gastric acid suppression can reduce the bio-
availability of posaconazole.31 Absorption is improved when the
total daily dose is divided into two to four doses.32 Therapeutic
drug monitoring is recommended in human patients; target peak
concentrations are greater than 1.48 mg/L.15,16 Posaconazole is
highly protein bound (>95%) and undergoes hepatic metabo-
lism, with some inhibition of P450 enzymes. Most administered
posaconazole is eliminated in the feces. Data on CSF concentra-
tions of posaconazole are not yet available but it appears to be
effective for treatment of CSF cryptococcosis. Adverse effects
are less common in cats when compared with voriconazole, but
information is limited at the time of writing.28-30,33
Other Azole Antifungals
Other azole drugs that are undergoing clinical trials in humans
are ravuconazole and isavuconazole. Ravuconazole is a flucon-
azole derivative with good oral bioavailability. Isavuconazole
is a water-soluble triazole with a spectrum similar to that of
posaconazole but is available in oral and IV formulations. It is
metabolized by P450 enzymes and does not achieve therapeutic
concentrations in the urine. The CNS penetration of isavucon-
azole has not yet been evaluated.34
Amphotericin B
Mechanism of Action and Spectrum of Activity
Amphotericin B (AMB) is a polyene macrolide antibiotic pro-
duced by Streptomyces nodosus. It is closely related to nystatin.
AMB irreversibly binds sterols in fungal cell membranes, form-
ing pores or channels with subsequent leakage of ions. Although
generally considered to be fungistatic, at high doses it may be
fungicidal. AMB also possesses immunomodulatory effects; it
activates macrophages and enhances macrophage-killing capac-
ity. This may help explain its efficacy against pathogens such as
Pythium insidiosum, which lack cell wall ergosterol.
Amphotericin B has antifungal activity against all of the
important small animal fungal pathogens. It is also active
against Leishmania spp.35,36 In human medicine, AMB is the
preferred drug for treatment of rapidly progressive mycoses,
92 SECTION 2  Antiinfective Therapy

BOX 9-1
Suggested Amphotericin B Infusion Protocols for Dogs and Cats

Formulation Protocol
All formulations Obtain baseline CBC, kidney panel, and UA and ensure adequate hydration before starting treatment.
Recheck a kidney panel before each infusion. Administer three times weekly for 4 weeks or until azotemia is
detected. For some patients, monthly administration of a single dose may be required to maintain remission.
Amphotericin B Reconstitute vial contents in sterile water to 5 mg/mL. Administer 0.9% NaCl IV at 1.5-2 times the cal-
deoxycholate (IV culated maintenance rate for 1 hour before and after AMB treatment. Ensure lines are flushed with D5W
route) before infusing AMB.
Dogs: Transfer 0.5 mg/kg to a 250-mL to 1000-mL bag of D5W (total fluid volume administered should
be based on body weight and ability to tolerate a fluid load). Administer IV over 4-6 hours.
Cats: Dilute 0.25 mg/kg in 30 mL of D5W and administer IV over 30 minutes to 1 hour.
Amphotericin B Transfer 0.5 mg/kg (cats) or 0.8 mg/kg (dogs) to bag containing 400 mL (cats) or 500 mL (dogs) of
deoxycholate (SC 0.45% sodium chloride in 2.5% dextrose. Administer subcutaneously. Fractious cats may need to be
route) sedated or placed in a restraint bag. Sterile injection site abscess formation may occur with this protocol,
especially with concentrations of amphotericin B > 20 mg/L.
Amphotericin Dilute to 1 mg/mL in D5W. Administer calculated dose IV over 1 to 2 hours.
B lipid complex Dogs: 3 mg/kg
(Abelcet) Cats: 1 mg/kg

AMB, Amphotericin B; D5W, 5% dextrose in water; UA, urinalysis.

immunosuppressed hosts, or fungal meningitis. Resistance to
AMB is rare, although high minimum inhibitory concentrations
have been observed for some molds, including some isolates of
Aspergillus terreus, Paecilomyces spp., and Scedosporium spp.,
as well as Sporothrix schenckii. The mechanism of resistance
is not well understood, but it may relate to decreased cell wall
ergosterol content.37
Clinical Use
Amphotericin B has poor aqueous solubility and is not absorbed
from the gastrointestinal tract, so it is formulated for IV infusion
in lyophilized form as a complex with the bile salt deoxycholate
(Fungizone, AMB-D) (Box 9-1). The complex forms a colloid in
water. Addition of electrolytes to the solution causes it to aggre-
gate, so it is administered in 5% dextrose (D5W). It is not neces-
sary to protect the infusion from light, as recommended in the
past. In the bloodstream, AMB dissociates from deoxycholate and
binds extensively to plasma proteins and cholesterol in membranes
throughout tissues. Penetration of the vitreous humor and CSF is
poor, but some animals with CNS infections still respond to treat-
ment. In human patients, AMB-D has been given intrathecally to
treat fungal meningitis and intraocularly following vitrectomy.38
A protocol for subcutaneous administration of AMB-D has
been reported for treatment of cryptococcosis,39 which may be
less costly than IV administration. Sterile injection site abscesses
occur in some animals treated using this protocol, so IV admin-
istration is preferred if possible.
Adverse Effects
The major adverse effect of AMB-D is nephrotoxicity, which is
dose dependent and transient if it is detected early and the drug
is discontinued. Both an acute effect caused by renal vasocon-
striction and a chronic cumulative effect from repeated admin-
istration occurs. Renal tubular acidosis, nephrogenic diabetes
insipidus, hypokalemia, and hypomagnesemia uncommonly
occur with treatment. Rarely, aggressive IV fluid therapy and
potassium supplementation to counteract these may be required.
Loading with sodium before the infusion may decrease nephro-
toxicity.40 Slow administration in a large volume of fluid also
decreases nephrotoxicity. Concurrent use of other nephrotoxic
drugs, such as aminoglycosides, should be avoided. In humans,
AMB-D can also cause fever, chills, headache, nausea, vomit-
ing, and, rarely, cytopenias and anaphylaxis.40 Fever, inappe-
tence, and vomiting also appear to occur in some dogs treated
with AMB-D. Phlebitis can occur at the IV infusion site. Other
adverse effects noted in humans include infusion-related hypo-
tension and hypochromic, normocytic anemia. NSAIDs can be
administered to decrease pyrexia during treatment.
Lipid Formulations of Amphotericin B
Three lipid formulations of AMB are marketed in the United
States. These are less nephrotoxic than AMB-D but are consid-
erably more expensive. Decreased nephrotoxicity results from a
reduced rate of transfer of AMB to mammalian cell membranes
and increased drug clearance from the blood by the mononu-
clear phagocyte system. This allows administration of a higher
dose of the drug, sometimes with improved treatment efficacy.
Nevertheless, renal function should still be monitored.
Amphotericin B colloidal dispersion (ABCD, Amphotec)
contains AMB and cholesterol sulfate, which form disc-shaped
particles. It is more likely to cause chills, fever, and hypotension
in humans than AMB-D (80% compared with 12% for AMB-
D), so it is given over 3 to 4 hours and with premedication.
Liposomal amphotericin B (Ambisome) is a widely used for-
mulation for treatment of human patients. It consists of AMB
and a lipid mixture (phosphatidylcholine, cholesterol, and dis-
tearoyl phosphatidylglycerol). After reconstitution in D5W, it
forms 80-nm vesicles. Nephrotoxicity and infusion-related reac-
tions are much less common with liposomal amphotericin B than
with ABCD or AMB-D, and the drug appears to reach higher
concentrations in the eye.41 In human patients, it is used as sal-
vage therapy for aspergillosis, cryptococcosis, and candidiasis.

TABLE 9-3
Suggested 5-Flucytosine Drug Doses for Cats
Drug Dose (mg/kg) Interval (hours) Species
5-Flucytosine 25 to 50 6 to 8 C PO
C, Cats.
Amphotericin B lipid complex (ABLC, Abelcet) is a mix-
ture of AMB, dimyristoyl phosphatidylcholine, and dimyris-
toyl phosphatidylglycerol that forms ribbon-like sheets. ABLC
is the most common formulation used in veterinary medicine.
In humans, nephrotoxicity and infusion-related reactions are
intermediate between AMB-D and liposomal amphotericin B.
ABLC has been used in small animals to treat refractory cryp-
tococcosis and cryptococcal meningitis and disseminated coc-
cidioidomycosis, aspergillosis, blastomycosis, histoplasmosis,
and pythiosis.
AMB cochleates are phosphatidylserine-calcium precipitates
in the form of a continuous lipid bilayer sheet rolled up in a
spiral. This formulation permits oral administration of AMB.
Cochleates have been effective when used to treat mouse mod-
els of candidiasis and aspergillosis without significant toxicity.
They remain under investigation on a research basis.
5-Flucytosine
Mechanism of Action and Spectrum of Activity
Flucytosine is a fluorinated pyrimidine related to fluorouracil.
It has activity only against Cryptococcus spp. and Candida
spp. These fungi deaminate flucytosine to 5-fluorouracil, which
interferes with DNA replication and protein synthesis. Mam-
malian cells cannot convert flucytosine into 5-fluorouracil, so
toxicity to mammalian cells is limited. Marked drug resistance
arises during treatment (secondary drug resistance), and so flu-
cytosine must always be used in combination with other drugs,
most commonly AMB. Flucytosine may be synergistic with
AMB. Resistance results from modifications in fungal enzymes
that are required for flucytosine uptake and metabolism.37
Clinical Use
Flucytosine is absorbed rapidly and well from the gastrointesti-
nal tract and is minimally bound to plasma proteins. Penetration
of the CSF and aqueous humor is excellent. In cats, flucytosine
may be considered to treat severe or refractory cryptococcosis
in combination with other agents. Because about 80% of the
dose is excreted unchanged in the urine, high urinary concentra-
tions can be achieved. For the same reason, flucytosine toxicity
is greatly increased in animals with renal failure, so the drug
should be avoided in these animals or the dose lowered and
plasma drug concentrations monitored carefully (Table 9-3).
In human patients, toxicity is correlated with 2-hour postpill
concentrations greater than 100 mg/L, and concentrations of
30 to 80 mg/L are suggested for treatment of cryptococco-
sis.15 Because AMB can cause impaired renal function, careful
monitoring for toxicity is indicated when flucytosine is used in
conjunction with AMB. The use of flucytosine in animals has
mostly been limited by its high cost.
CHAPTER 9  Antifungal Drugs 93
Route Comments
Monitor CBC. Use cautiously in animals with impaired
renal function. Avoid use in dogs. Serum concentra-
tion monitoring is recommended in humans.
Adverse Effects
Administration of flucytosine should be avoided in dogs, which
often develop a severe (but reversible) drug eruption within 2
to 3 weeks of starting treatment. The most common adverse
effects of flucytosine in cats are myelosuppression and gastroin-
testinal signs. The CBC should be monitored during treatment,
and therapeutic drug monitoring should be considered. Myelo-
suppression is more likely to occur in human patients receiving
other myelosuppressive drugs. Other adverse effects in humans
include rash and reversible increases in liver enzyme activities.
Flucytosine toxicity may result from conversion of flucytosine
to 5-fluorouracil by the intestinal microflora.
Griseofulvin
Mechanism of Action and Spectrum of Activity
Griseofulvin is an oral antifungal drug derived from Penicillium
griseofulvum that binds to fungal tubulin, leading to impaired
microtubule function and mitotic arrest. It also has antiinflam-
matory properties.42 After administration, griseofulvin is rap-
idly deposited in keratin precursor cells in the skin and hair, but
disappears from the stratum corneum within 48 to 72 hours of
discontinuation of treatment. Griseofulvin is fungistatic and has
a limited spectrum of activity. It is used primarily to treat der-
matophytosis and is not effective against yeasts such as Candida
spp. and Malassezia spp. Resistance has been documented in
some dermatophytes and may be due to altered tubulin.
Clinical Use
The absorption of griseofulvin is increased when it is adminis-
tered with a fatty meal or whole milk. The drug is available in
microsized and ultramicrosized formulations, which improve
absorption from the stomach and small intestine. Most veterinary
preparations are microsized. The dose of the ultramicrosized for-
mulation can be reduced by 50%, because of improved absorp-
tion, but efficacy remains unchanged (Table 9-4). Prolonged
treatment periods (months) are required for dermatophytosis (see
Chapter 58).43
Adverse Effects
Griseofulvin is a potent inducer of cytochrome P450 enzymes
and so decreases the efficacy of other drugs that are metabolized
to inactive metabolites by P450 enzymes. It is teratogenic, so
it should not be used to treat pregnant animals. Other adverse
effects of griseofulvin include inappetence, vomiting, and diar-
rhea. In cats, it can cause myelosuppression, which is generally
reversible on discontinuation of treatment, but irreversible pan-
cytopenia has been reported.44 Myelosuppression may be more
likely to occur in cats with FIV infection.45 The CBC should be
monitored every 2 weeks during treatment.
94 SECTION 2  Antiinfective Therapy
TABLE 9-4
Suggested Griseofulvin and Terbinafine Doses for Dogs and Cats
Drug Dose (mg/kg) Interval (hours) Species Route Comments
Griseofulvin 25 12 D, C
(microsized)
Griseofulvin 15 12 D, C
(ultramicrosized)
Terbinafine 30 to 40 24 D, C
hydrochloride
C, Cats; D, dogs.
Terbinafine
Mechanism of Action and Spectrum of Activity
Terbinafine is a synthetic allylamine that inhibits fungal squa-
lene epoxidase, which blocks fungal lanosterol and ergosterol
synthesis and leads to accumulation of toxic squalene, with
resultant fungal cell lysis. It is most effective for treatment of
dermatophytosis. It has also been used to treat Malassezia spp.
dermatitis in dogs.46 The efficacy of terbinafine for treatment
of invasive fungal infections has not been well investigated.
It has been used both successfully47 and unsuccessfully48 in
combination with other drugs to treat pythiosis in dogs, and
as an alternative to itraconazole to treat human sporotri-
chosis.49 It also has in vitro activity against other dimorphic
fungi and molds, although its activity is highest for dermato-
phytes.50,51 Terbinafine may be synergistic when administered
with other antifungal drugs, which may provide enhanced
efficacy for treatment of refractory mycoses.52 However, non-
dermatophyte molds including Aspergillus spp. and Fusarium
spp. have been detected in people with onychomycosis that
failed to respond to terbinafine treatment.53 Resistance has
been reported in dermatophytes as a result of altered squalene
epoxidase.54
Clinical Use
Terbinafine is available in tablet form (Lamasil). It is well
absorbed in dogs when given with food (see Table 9-4).55 The
drug is highly lipophilic and accumulates in skin, claws, hair,
and fat, where it persists for weeks after treatment is discontin-
ued. Hepatic and renal failure increase plasma levels unpredict-
ably, and dosage reduction may be required.
Adverse Effects
Terbinafine is generally well tolerated. Gastrointestinal signs,
especially vomiting and increased liver enzyme activities, are
uncommon. Facial pruritis has been reported in cats.56
Echinocandins
The echinocandins are lipopeptide antifungals that inhibit the
formation of β-1,3-d-glucans in the fungal cell wall, a mecha-
nism of action that is completely different from that for AMB
PO Administration with fatty food improves absorption.
Dose may be increased to 50 mg/kg q12h for refrac-
tory infections. Avoid in cats with FIV infections.
Do not use in pregnancy. Drug interactions pos-
sible. Monitor CBC.
PO Avoid in cats with FIV infections. Do not use in preg-
nancy. Drug interactions possible. Monitor CBC.
PO Administer with food.
and the azoles. The prototype drug is caspofungin acetate (Can-
cidas). Other drugs in this class are micafungin and anidula-
fungin. Caspofungin is fungicidal against Candida spp. and
fungistatic against Aspergillus species. Treatment with caspo-
fungin induced remission in a dog with disseminated aspergil-
losis (1 mg/kg IV in 250 mL 0.9% NaCl over 1 hour, q24h).25
Synergism has been reported when echinocandins are used in
combination with other antifungal drugs.57 The echinocandins
are ineffective against Cryptococcus spp., which possess little
glucan synthase, and were only effective for treatment of Coc-
cidioides spp. in a mouse model when used in conjunction with
AMB.58 In human patients, caspofungin is approved for treat-
ment of refractory invasive aspergillosis, but it is also effective
for mucosal candidiasis.
Caspofungin is given once daily as a slow IV infusion. In
human patients, it is extensively protein bound and metabolized
slowly by the liver, with some renal excretion. It has limited
ability to penetrate the CNS and eye.59,60 The cost is similar to
that of lipid formulations of amphotericin B. Adverse effects
noted in humans include fever, phlebitis, and increased activity
of serum ALT (<20% of patients). The proper dose and extent
to which adverse effects occur in dogs and cats is unknown, but
administration of anidulafungin to the dog treated with caspo-
fungin was associated with the development of a severe diffuse
urticarial reaction.25
Other Antifungal Treatments
In human patients, hyperbaric oxygen therapy has been used
to treat some fungal infections in combination with antifungal
drugs. Whether this is beneficial or harmful is controversial and
requires further study.61
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Hope WM, Billaud EM, Lestner J, et al. Therapeutic drug monitoring
for triazoles. Curr Opin Infect Dis. 2008;21:580-586.
Pound MW, Townsend ML, Dimondi V, et al. Overview of treatment
options for invasive fungal infections. Med Mycol. 2011;49:561-580.

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CHAPTER 10
Antiprotozoal Drugs
Jane E. Sykes and Mark G. Papich
w KEY POINTS
• A  ntiprotozoal drugs often have a restricted spectrum of activ-
ity, although some are also active against bacteria and fungi.
Many interfere with enzyme pathways specific to certain proto-
zoal species.
• Treatment with antiprotozoal drugs may not consistently clear
an infection.
INTRODUCTION
The use of antiprotozoal drugs in dogs and cats is frequently
extrapolated from their use in human patients or food animal spe-
cies. Almost all antiprotozoal drugs are not specifically approved
for treatment of protozoal infections in dogs and cats. Some
antibacterial and antifungal drugs also have antiprotozoal activ-
ity (see Chapters 8 and 9 for the use and adverse effects of these
drugs). In vitro activity of antiprotozoal drugs, and monitoring of
resistance, is more difficult for antiprotozoal drugs because stan-
dardized susceptibility testing is not routinely performed for these
pathogens. In addition, many antiprotozoal drugs are designed to
be active in the lumen of the intestine for treatment of intestinal
protozoal infections and the concentration of active drug in the
intestinal lumen after oral administration is difficult to measure.
Therefore, the concentration of drug to which these pathogens are
exposed is often not known. The activity and dosage regimens of
antiprotozoal drugs are often based on the results of clinical trials,
rather than concentration-exposure relationships between anti-
protozoal drugs and the organism of interest.
Many antiprotozoal drugs are active only against a restricted
range of protozoal species. To reflect this, drugs in this chapter are
organized into antiprotozoal drugs primarily used for gastroin-
testinal infections; those with a broad spectrum of activity; those
used to treat systemic protozoal infections (such as hepatozoono-
sis, toxoplasmosis, neosporosis, and sarcocystosis); antiprotozoal
drugs used to treat leishmaniosis; and those used to treat Chagas’
disease. For many infections, treatment does not consistently
result in parasitologic cure.
Antiprotozoal Drugs Used Primarily
for Gastrointestinal Infections
Amprolium
Amprolium is a thiamine analogue that is used to prevent and
treat intestinal coccidiosis (Table 10-1). It is available as a feed
additive for livestock and is sometimes administered in food
or drinking water to puppies and kittens. Adverse effects of
anorexia or diarrhea are rare and primarily occur at high doses
and with prolonged use. Central nervous system (CNS) signs
• R  esistance to antiprotozoal drugs is a growing problem among
protozoal pathogens.
• Some antiprotozoal drugs have minimal to no adverse effects,
whereas for others, adverse effects severely limit use.
• The availability of antiprotozoal drugs is restricted in some
countries.
can result from thiamine deficiency, which is reversible on addi-
tion of thiamine to the diet. However, thiamine supplementa-
tion may interfere with the drug’s efficacy.
Benzimidazoles
Fenbendazole and Albendazole
Benzimidazoles bind to β-tubulin within a variety of helminths
and protozoa. This leads to inhibition of tubulin polymerization
and the formation of microtubules, with impaired cell division.
Glucose uptake by parasites is also impaired. Resistance can
result from production of altered β-tubulin by parasites, which
reduces binding of benzimidazole drugs.
Fenbendazole is widely used to treat giardiasis in dogs and
cats. It is safer than metronidazole, can be administered to young
animals, and has higher efficacy, although treatment failure can
still occur. A second course of treatment or administration of
fenbendazole in combination with metronidazole can be effec-
tive in refractory cases. Administration with food may improve
absorption, but the fat content of the food does not influence
absorption.1 Adverse effects of fenbendazole are very rare but
can include decreased appetite, vomiting, diarrhea, and rarely
reversible pancytopenia.2 At high doses used to treat Mesoces-
toides spp. peritonitis (100 mg/kg q12h), neurologic signs have
been observed.3 Febantel is metabolized to a benzimidazole
compound and has been used in combination with praziquantel
and pyrantel (Drontal Plus) to treat Giardia spp. infection in
dogs, although efficacy at label dosages has been variable and
some dogs can re-shed low numbers of cysts when treatment
is discontinued.4 Albendazole has an affinity for rapidly divid-
ing cells, and although it is used extensively for treatment of
parasitic infections in human patients, it been associated with
anorexia and reversible bone marrow suppression in dogs and
cats especially when high doses are administered for more than
5 days.5 As a result, fenbendazole is used more commonly in
small animals.
Nitroimidazoles
Protozoa reduce nitroimidazoles to nitro anion free radicals,
which cause damage to parasite DNA. Some nitroimidazoles are
mutagens and carcinogens, but carcinogenesis has not been dem-
onstrated in dogs and cats with long-term use. Metronidazole,
97
98 SECTION 2  Antiinfective Therapy

TABLE 10-1
Suggested Doses of Drugs That Are Primarily Used to Treat Protozoal Infections of the Gastrointestinal Tract in Small Animals
Dose Interval Duration
Drug (mg/kg) (hours) Species Route (days) Comments
Fenbendazole 50 24 C, D PO 5 Giardiasis. May be administered with
food. Safe in pregnancy.
Albendazole 25 12 C, D PO 3 Giardiasis. May cause bone marrow sup-
pression. Do not use in pregnancy.
Metronidazole 15 12 C, D PO 8 Giardiasis. Use caution with hepatic
insufficiency. Dose for metronidazole
benzoate is 25 mg/kg.
Tinidazole 15 12 D PO 5 Giardiasis. Administer with food or in
24 C capsules to reduce bitterness.
Ronidazole 30 24 C PO 14 Tritrichomoniasis. Avoid doses ≥60 mg/
kg/day. Compounded from powder.
Paromomycin 10 8 D PO 5-10 Cryptosporidiosis. Caution in animals
with diarrhea due to possible systemic
absorption. Avoid in cats.
Nitazoxanide 100 mg/animal 12 D, C PO 3 Cryptosporidiosis. Efficacy and safety
unclear. Vomiting common in cats.
Amprolium 1.25 g of 20% 24 D, C PO 7 Isosporiasis. Add to food. Do not ad-
powder or 30 mL minister for prolonged periods.
of 9.6% solution
to 3.8 L of water
Sulfadimethoxine 55 on day 1, 27 24 D, C PO 3-23 or for 48 hr Isosporiasis with or without a dihydro-
thereafter after signs resolve folate reductase inhibitor.

C, Cats; D, dogs.

ronidazole, and tinidazole have primarily been used to treat
enteric protozoal infections. Benznidazole is specifically used to
treat infections with Trypanosoma cruzi.
Metronidazole
Metronidazole is used to treat giardiasis in dogs and cats,
although efficacy may be as low as 50%. It also has activity
against amoebic infections. The clinical use and adverse effects
of metronidazole are described in Chapter 8. Doses of metro-
nidazole used for treatment of giardiasis have the potential to
be associated with neurotoxicity, so fenbendazole is preferred
because of greater safety and efficacy. Metronidazole can be
combined with fenbendazole for refractory giardiasis.
Tinidazole
Tinidazole is a 5-nitroimidazole that has amoebicidal, giardicidal,
trichomonicidal, and anaerobic bactericidal activity. It is some-
times used as a single-dose treatment for giardiasis in human
patients. The efficacy of tinidazole for treatment for giardiasis in
dogs and cats has not been evaluated, and the half-life in dogs (4.4
hours) and cats (8.4 hours) is shorter than that in human patients
(>12 hours).6,7 Tinidazole is very well absorbed in dogs and cats,
with a bioavailability of 100%. Adverse effects are similar to those
of metronidazole. Like metronidazole, tinidazole has a bitter taste.
Ronidazole
Ronidazole is the drug of choice for treatment of Tritrichomonas
foetus infections, which are less responsive to metronidazole
and tinidazole.8-10 Resistance to ronidazole has been identified
in some isolates of T. foetus and is associated with treatment
failure in infected cats.11 Resistance is thought to result from
increased oxygen-scavenging capacity by the parasite, whereby
oxygen competes effectively with ronidazole and other nitro-
imidazoles for ferredoxin-bound electrons.
Ronidazole is absorbed rapidly and completely after oral
administration to cats. Some compounded formulations may
have decreased efficacy as a result of low ronidazole content or
differences in drug release at the site of action (the large bowel).
A modified-release formulation that is delivered to the colon
may have improved efficacy.12 Decreased appetite, vomiting,
and neurologic signs can occur in dogs and cats, especially at
doses above 30 mg/kg q12h in cats and at doses as low as 10 mg/
kg/d in dogs.13 Once daily dosing is probably sufficient because
of the long half-life of the drug in cats.12 Doses of 20 mg/kg or
less may not effectively clear infection with T. foetus. Neuro-
logic signs result from γ-aminobutyric acid (GABA) antagonism
in the CNS and include ataxia, decreased mentation, agitation,
tremors, and hyperesthesia, which occur up to 9 days after the
start of treatment and resolve when the drug is discontinued.13
Nitozoxanide
Nitazoxanide is a nitrothiazolyl-salicylamide derivative that has
activity against Giardia spp., Cryptosporidium spp., Sarcocystis
neurona, some anaerobic bacteria, Helicobacter spp., and Cam-
pylobacter jejuni. It inhibits the pyruvate-ferredoxin/flavodoxin
oxidoreductase enzyme-dependent electron transfer reaction

that is essential for anaerobic metabolism in these organisms.
Resistance has been documented in Giardia spp.14
Reports of nitazoxanide use in dogs and cats have been rare,
and its efficacy in dogs and cats is largely unknown. An equine
formulation (Navigator) that was used to treat equine proto-
zoal meningoencephalitis caused by Sarcocystis neurona has
been removed from the market. Doses have been extrapolated
from those used for human patients. Nitazoxanide treatment of
cats co-infected with Cryptosporidium spp. and T. foetus led to
cessation of shedding during treatment, but infection was not
eliminated.15 Vomiting occurred frequently, especially at higher
doses (75 mg/kg PO q12h). In humans, nitazoxanide is rapidly
absorbed from the gastrointestinal tract and metabolized to the
active metabolite tizoxanide, which is highly protein bound.
After hepatic glucuronidation, it is excreted in urine and bile.
Antibacterial Drugs with Broad-Spectrum
Antiprotozoal Activity
Folic Acid Antagonists
Trimethoprim, pyrimethamine, ormetoprim, and sulfadiazine
inhibit parasite replication through folate antagonism. Synergis-
tic combinations of sulfadiazine with trimethoprim or pyrimeth-
amine are primarily used to treat toxoplasmosis, neosporosis,
and intestinal coccidiosis (Isospora spp. infections) in dogs and
cats. A combination of pyrimethamine, trimethoprim-sulfadia-
zine, and clindamycin has also been used to treat Hepatozoon
americanum infections.16 The mechanisms of action, use, and
adverse effects of trimethoprim and sulfonamides are discussed
in Chapter 8.
Pyrimethamine
Like trimethoprim, pyrimethamine inhibits dihydrofolate reduc-
tase, which is necessary for synthesis of thymidine. However, in
contrast to trimethoprim, it has a greater affinity for the pro-
tozoal enzyme than the bacterial enzyme. Resistance to pyri-
methamine can occur when parasites synthesize dihydrofolate
reductase enzymes with an altered drug target site. Pyrimeth-
amine is well absorbed after oral administration and penetrates
a variety of tissues including the CNS. Hepatic metabolism and
some renal excretion occur. Although clearance of pyrimeth-
amine is not affected by renal disease, the use of caution with
hepatic or renal insufficiency has been recommended in human
patients.
Pyrimethamine is well tolerated. Gastrointestinal signs such
as vomiting, diarrhea, and decreased appetite occur in some
treated animals. Bone marrow suppression can occur with pro-
longed treatment at higher doses as a result of folic acid defi-
ciency. In human patients, concurrent administration of folinic
acid is recommended when high doses are used for treatment
of toxoplasmosis. Folinic acid, but not folic acid supplemen-
tation also reverses marrow suppression in dogs treated with
pyrimethamine.17 The CBC should be monitored weekly during
treatment, and supplementation should be provided if leukope-
nia develops and continued treatment is necessary. Stomatitis,
ulcerative glossitis, and exfoliative dermatitis have also been
described in human patients as a result of folic acid deficiency.18
Other adverse effects of pyrimethamine-sulfadiazine combina-
tions result from the sulfadiazine component (see Chapter 8).
Unlike trimethoprim-sulfadiazine, there are no approved
formulations of pyrimethamine-sulfonamides for dogs and
CHAPTER 10  Antiprotozoal Drugs 99
cats. Pyrimethamine is available as a single agent in tablets but
should be administered with a sulfonamide for the best efficacy.
Another alternative is the combination of pyrimethamine-sulfa-
diazine, which is available in an oral liquid suspension for horses
(ReBalance). Although off-label, it is a convenient formulation
for small animal veterinarians. This formulation can be admin-
istered at a dose of 1 mg/kg pyrimethamine + 20 mg/kg sulfa-
diazine PO q24h. This is equivalent to 0.33 mL of the equine
formulation per 4 kg of body weight for dogs and cats.
Macrolides and Lincosamides
Clindamycin, azithromycin, and clarithromycin have antipro-
tozoal activity. The use of these macrolides and lincosamides in
dogs and cats and their adverse effects are discussed in Chap-
ter 8. Clindamycin is the most widely used antiprotozoal for
treatment of toxoplasmosis and neosporosis in dogs and cats.
Although clindamycin inhibits shedding of Toxoplasma gondii
oocysts by cats,19 clinical efficacy of clindamycin for treating
toxoplasmosis in dogs and cats has been questioned by experts
and in published studies. Trimethoprim-sulfonamides are a suit-
able alternative, or if clindamycin is used, pyrimethamine may
be used in combination. In human patients, pyrimethamine and
clindamycin are used as a substitute for pyrimethamine and
sulfadiazine for treatment of toxoplasmosis in sulfadiazine-
sensitive individuals. Azithromycin is used in combination with
atovaquone for treatment of babesiosis and cytauxzoonosis.
Paromomycin
Paromomycin is the only aminoglycoside antibiotic that has
efficacy against protozoa. It is poorly absorbed from the gas-
trointestinal tract and so has been used to treat enteric proto-
zoal infections, particularly cryptosporidiosis. It is ineffective
for treatment of tritrichomoniasis in cats.10 Furthermore, when
used to treat intestinal protozoal infections in cats, paromomy-
cin has been absorbed systemically because of intestinal muco-
sal compromise, with resultant acute renal failure, deafness,
and cataract formation.20 As a result, its use has been limited.
In human patients, paromomycin has been used topically to
treat cutaneous leishmaniasis and parenterally to treat visceral
leishmaniasis.21
Tetracyclines and Ciprofloxacin
Doxycycline has primarily been used for malaria prophylaxis in
humans. Ciprofloxacin is thought to inhibit DNA gyrase within
a chloroplast organelle (the apicoplast) of apicomplexan para-
sites (see the triazines, later). It is an alternative to sulfadiazine
for treatment of isosporiasis in human patients.18 Tetracyclines
and ciprofloxacin have not been widely used for prevention or
treatment of protozoal infections in dogs and cats with the pos-
sible exception of doxycycline as part of combination treatment
for babesiosis (see Chapter 75).
Antiprotozoal Drugs Used for Systemic
Protozoal Infections
Quinolone Derivatives
Atovaquone
Atovaquone is a hydroxynaphthoquinone that inhibits electron
transport in protozoa by targeting the cytochrome bc1 com-
plex (Table 10-2). It has been used in combination with other
antiprotozoal drugs as an alternative treatment for malaria,
100 SECTION 2  Antiinfective Therapy

TABLE 10-2
Suggested Doses of Drugs Primarily Used to Treat Systemic Protozoal Diseases Excluding Leishmaniosis and Trypanosomiasis
in Small Animals
Dose Interval Duration
Drug (mg/kg) (hours) Species Route (days) Comments
Pyrimethamine 1 24 D PO 14-28 Primarily neosporosis, toxoplasmosis, and American hep-
0.5-1 C atozoonosis. Use with a sulfonamide. Use caution with
hepatic and renal insufficiency. Monitor CBC. Folinic
acid supplementation (5 mg/day) may be required.
Clindamycin 22 12 D, C PO Toxoplasmosis, neosporosis, sarcocystosis, and
Americ­an hepatozoonosis.
Azithromycin 10 24 D, C PO 10 Babesiosis and cytauxzoonosis. Used with atovaquone.
Atovaquone 13.3 8 D PO 10 Babesiosis and cytauxzoonosis with azithromycin.
15 C Administer with food.
Decoquinate 10-20 12 D PO ≥365 American hepatozoonosis and sarcocystosis. Powder
(6% decoquinate; 60 mg active ingredient per gram)
is mixed with food. This equates to 0.5 to 1 table-
spoon/10 kg body weight q12h.
Imidocarb 6.6 Once, D Deep IM N/A Large Babesia spp. infections. Caution with hepatic or
dipropionate 5 repeat in C renal insufficiency. Avoid use with other cholinesteras­e
14 days inhibitors.
Diminazene 3-5 Once D Deep IM N/A Babesiosis and African trypanosomiasis. Narrow
aceturate therapeutic range.
Ponazuril 20-50 12-24 D PO 3-28 Toxoplasmosis, neosporosis, isosporiasis. Optimal dose,
duration, efficacy, and adverse effects unknown.
Toltrazuril 5-10 12-24 D PO 1-14 Hepatozoonosis, isosporiasis. One dose may be effec-
18 C tive for isosporiasis. Optimal dose and duration for
hepatozoonosis unknown.

C, Cats; D, dogs; N/A, not applicable.

toxoplasmosis, and pneumocystosis in human patients.18 In
veterinary medicine, atovaquone is used in combination with
azithromycin for treatment of Babesia gibsoni and Babesia
conradae infections and cytauxzoonosis (see Chapters 75 and
76), but is expensive. Resistance has been reported in Plas-
modium spp., T. gondii, Pneumocystis jirovecii, and canine
B. gibsoni strains as a result of mutations in the cytochrome
bc1 complex.
Atovaquone is highly lipophilic and extensively protein
bound. Little is known about atovaquone metabolism in dogs
and cats. Its oral bioavailability increases significantly when food
is administered concurrently. Atovaquone is available alone or
in a formulation with proguanil for treatment and prevention of
malaria in human patients. The combination formulation may
cause vomiting and diarrhea in dogs, but otherwise atovaquone
appears well tolerated by both dogs and cats.22-24 In human
patients, adverse effects have included gastrointestinal signs,
headache, fever, and increased liver enzyme activities. Coadmin-
istration with metoclopramide, tetracycline, or rifampin signifi-
cantly decreases plasma drug concentrations in humans.
Decoquinate
Decoquinate is a 4-hydroxyquinolone coccidiostat that inhib-
its electron transport in protozoal mitochondria and interferes
with sporozoite development. It likely has a similar mechanism
of action to atovaquone, because resistance to atovaquone can
result in cross-resistance to decoquinate.25 Although developed
as a food additive for use in production animals, decoquinate
can be used successfully and without adverse effects to prevent
relapses in dogs that are chronically infected with Hepatozoon
americanum.16 Decoquinate also appeared to be effective for
treatment of Sarcocystis spp. myositis in a dog.26 Its distribution
and metabolism in dogs and cats have not been described.
Aromatic Diamines
The aromatic diamines include imidocarb dipropionate, dim-
inazene aceturate, pentamidine isethionate, and phenamidine
isethionate. These drugs inhibit DNA synthesis in protozoa. In
dogs, imidocarb and diminazene have been used most widely,
primarily for treatment of Babesia spp. and Hepatozoon canis
infections. In general, the use of imidocarb has been preferred
over diminazene because of diminazene’s narrow therapeu-
tic index. Pentamidine and phenamidine have also been used
in dogs. Pentamidine isethionate is a second-line treatment for
leishmaniasis in human patients and is used as an alternative
to diminazene for treatment of African trypanosomiasis (see
­Chapter 78). Treatment of protozoal infections using the aro-
matic diamines may not result in complete parasite elimination.
Imidocarb Dipropionate
Imidocarb dipropionate is primarily used to treat large Babesia
spp. and Hepatozoon canis infections in dogs. More effective

agents have replaced imidocarb for treatment of canine mono-
cytic ehrlichiosis and feline cytauxzoonosis.
Imidocarb is given by intramuscular injection, twice, 2 weeks
apart. It is generally well tolerated, although it can cause tran-
sient pain at the site of injection. It appears to be eliminated
in urine and feces.27 Acute adverse effects result from its anti-
cholinergic activity and include vomiting, shivering, hypersali-
vation, lacrimation, diarrhea, agitation, lethargy, pyrexia, and
periorbital swelling. These generally resolve within a few hours.
A possible association between imidocarb treatment and acute
renal tubular necrosis has been reported in dogs.28 Massive
hepatic necrosis was described after overdosage of dogs with 10
times the recommended dose.29
Diminazene Aceturate
Diminazene aceturate is administered parenterally, primarily to
dogs, for treatment of babesiosis, African trypanosomiasis, and,
most recently, infections with Rangelia vitalii, a novel protozoal
pathogen of dogs from Brazil.30 Diminazene aceturate is not
readily available in the United States. Resistance to diminazene
has been described in Babesia gibsoni.31
Although formulations for small animals are not available,
a powdered commercial drug formulation (Veriben) has been
reconstituted with sterile water to a concentration of 7 mg/mL
and administered intramuscularly at a dose of 3 mg/kg dim-
inazene diaceturate for a pharmacokinetic study in cats.32 The
dose was well tolerated, and diminazene was eliminated with
a half-life of only 1.7 hours and a peak concentration of only
0.5 µg/mL. Development of clinically effective doses from these
data requires further studies. Diminazene was administered to
seven cats experimentally infected with Trypanosoma evansi at
a dose of 3.5 mg/kg intramuscularly on five consecutive days.33
The treatment was 85.7% efficacious for elimination of the par-
asite, and no adverse effects were observed.
Effective doses of diminazene approach doses that are toxic,
so it should be used with caution.34 Adverse effects include
tachycardia and CNS signs such as ataxia, nystagmus, and opis-
thotonos. A single treatment can be given, or the dose can be
repeated 72 to 96 hours after drug administration. In some pro-
tocols for B. gibsoni infections, diminazene administration has
been followed a day later by treatment with imidocarb.
Triazine Antiprotozoals (Toltrazuril and Ponazuril)
Toltrazuril and its major metabolite ponazuril (toltrazuril sul-
fone, Marquis) are triazine-based antiprotozoal drugs that have
specific activity against apicomplexan coccidial infections. Tol-
trazuril is not available within the United States. Ponazuril and
toltrazuril appear to act on a plastid-like chloroplast organelle
(the apicoplast) in apicomplexan protozoa, which may have
originally been acquired from a green alga.35 This organelle
contains a small circular genome and operates key biochemi-
cal pathways. Although their mechanism of action is unclear,
triazines may inhibit metabolic enzymes or decrease pyrimi-
dine synthesis within the apicoplast. Ponazuril and toltrazuril
have been used in animals to treat isosporiasis, toxoplasmosis,
neosporosis, and equine protozoal meningoencephalitis. Tri-
azine resistance has been described in coccidia from production
animals.36
In dogs and cats, toltrazuril has been used to treat isospo-
riasis and hepatozoonosis, although infection may persist in
some animals.16,37-42 In Europe it is available in combination
with the anthelmintic emodepside (Procox Oral Suspension for
CHAPTER 10  Antiprotozoal Drugs 101
Dogs, Bayer Animal Health) for treatment of isosporiasis and
roundworm infections in puppies over 2 weeks of age. The sus-
pension contains 18 mg/mL of toltrazuril, and a single 9-mg/kg
dose is recommended. Toltrazuril appeared to be as effective
as trimethoprim-sulfadiazine-clindamycin-pyrimethamine for
treatment of canine American hepatozoonosis.16 One dog with
Hepatozoon canis infection recovered after treatment with tol-
trazuril and trimethoprim-sulfamethoxazole,42 but a toltrazuril
and imidocarb combination did not offer benefit over treat-
ment of H. canis infection with imidocarb alone.43 Dogs with
H. canis infection respond clinically within 72 hours of starting
treatment with toltrazuril. Anecdotal reports exist of the use of
ponazuril to treat Isospora spp. infections in dogs and cats, but
its efficacy is unclear. Doses of ponazuril used have ranged from
20 to 50 mg/kg for 2 to 5 days. In one dog, ocular toxoplasmo-
sis that was refractory to clindamycin resolved after treatment
with ponazuril for 28 days.44
The pharmacokinetics of the triazines in dogs has not been
reported. In horses, ponazuril crosses the blood-brain barrier to
some extent and achieves concentrations in the cerebrospinal
fluid sufficient to inhibit protozoa. It has long half-life in horses
(>4 days) and in cattle (2 to 3 days). Because of its specific activ-
ity against apicomplexan parasites, significant toxicity in mam-
malian species has not been reported.
Antileishmanial Antiprotozoal Drugs
Antifungal Agents
Amphotericin B
Amphotericin B is a treatment of choice for human visceral leish-
maniasis.18 It is thought to bind to ergosterol in the protozoal
membrane, and blocks the ability of Leishmania spp. to bind to
and enter macrophages.45 Lipid amphotericin B formulations have
been used to treat visceral leishmaniosis in dogs, although organ-
ism persistence and relapse have been reported in some dogs after
twice-weekly treatment for up to 10 administrations (0.8 to 3.3 mg/
kg intravenously).46,47 In an effort to reduce selection for resistant
parasites, the World Health Organization recommends against the
use of amphotericin B for treatment of canine leishmaniosis.48 A
full discussion of amphotericin B is provided in Chapter 9.
Azole Antifungals
Azole antifungals such as fluconazole have been used to treat
cutaneous leishmaniasis in human patients. More effective
antiprotozoal drugs are substituted for visceral leishmaniasis.
Posaconazole and ravuconazole also have activity against Try-
panosoma cruzi (see Chapter 78), and fluconazole may have
activity against Toxoplasma (see Chapter 72).
Antimony Compounds
Sodium stibogluconate (Pentostam) and N-methylglucamine
antimoniate (meglumine antimoniate, Glucantime) are derived
from the heavy metal antimony (Sb). They are called pentava-
lent antimony compounds (symbol Sbv) because they contain
Sb atoms that have five electrons in their outer shell. Pentava-
lent antimonials have been recommended as the first choice for
treatment of Leishmania infections in humans and in dogs.49
Their mechanism of action is still not completely clear, but it
is thought that pentavalent antimony undergoes reduction to
the more toxic trivalent version, possibly within macrophage
phagolysosomes or within the parasite itself.50 The trivalent
compound inhibits protozoal enzymes and damages protozoal
102 SECTION 2  Antiinfective Therapy
TABLE 10-3
Suggested Doses of Drugs Primarily Used to Treat Leishmaniosis or Chagas’ Disease in Dogs
Dose Interval Duration
Drug (mg/kg) (hours) Route (days)
Meglumine 75-100 24 SC 30-60
antimoniate
Allopurinol 10 12 PO At least Leishmaniosis. Use with meglumine or miltefosine. Monitor
6-12 months
Miltefosine 2 24 PO 28
Benznidazole 5-10 24 PO 60
DNA.51 Resistance to antimonials is an emerging problem in
human Leishmania spp. isolates. This has led to the use of
higher drug dosages, with an associated rise in the rate of drug
toxicity. The availability of antimonial drugs is limited in the
United States.
Both sodium stibogluconate and meglumine antimoniate are
administered subcutaneously (Table 10-3). The treatment of
choice for canine leishmaniosis is a combination of meglumine
antimoniate and allopurinol.48,49 Complete clearance of the infec-
tion may not always occur.52 After subcutaneous administration,
more than 80% of meglumine antimoniate is eliminated by the
kidneys. Pain at the site of injection is the most common adverse
effect, and cutaneous abscesses or cellulitis may also occur.53
Systemic adverse effects such as lethargy, vomiting, diarrhea,
inappetence, and increased serum liver enzyme activities may be
more likely to occur in the presence of renal failure, which can be
a complication of leishmaniosis. Of concern, the administration
of meglumine antimoniate to healthy dogs has led to renal tubu-
lar necrosis, in the absence of azotemia.54 In human patients,
other adverse effects include generalized arthralgias, abdominal
pain, mild cytopenias, cardiotoxicity, and chemical pancreati-
tis.21 Novel formulations of antimony compounds are being
developed for treatment of leishmaniasis, including lipid formu-
lations and preparations that could be administered orally.50
Allopurinol
Allopurinol is a purine analogue. In parasites, allopurinol is metab-
olized to derivatives that are incorporated into RNA, which leads
to impaired protein synthesis. Resistance can occur and appears to
result from reduced activity of purine transporters and a reduced
ability to accumulate purine.55 Although it has been used alone for
treatment of canine leishmaniosis, allopurinol is most commonly
used in combination with meglumine or miltefosine.
Allopurinol is rapidly absorbed from the gastrointestinal tracts
of dogs. Peak drug concentrations occur 1 to 3 hours after adminis-
tration.56 Allopurinol is not bound to plasma proteins. Allopurinol
rarely causes adverse effects in dogs. It inhibits mammalian xan-
thine oxidase, which results in decreased uric acid production from
xanthine. As a result, long-term use can result in xanthine urolithi-
asis.57 In human patients, allopurinol most commonly causes skin
rashes. Gastrointestinal signs can also occur. Rarely administra-
tion of allopurinol to humans results in aplastic anemia or a hyper-
sensitivity syndrome characterized by toxic epidermal necrolysis
with hepatic and renal failure.58,59 Serious adverse reactions are
Comments
Leishmaniosis. Use with allopurinol. Available in ampules. Dose
based on concentration of meglumine antimoniate. Caution
in dogs with renal insufficiency.
CBC and serum chemistry in dogs with hepatic and renal
insufficiency.
Leishmaniosis. Use with allopurinol.
Chagas’ disease
more likely to occur in human patients with renal insufficiency. At
usual doses, allopurinol treatment leads to improvement in kidney
lesions in dogs with leishmaniosis that have renal insufficiency.60
Nevertheless, careful monitoring for adverse effects is indicated in
these dogs (see Table 10-3). Serious drug interactions can occur
when allopurinol is administered with azathioprine.
Miltefosine
Miltefosine is an effective alternative to pentavalent antimonials
for treatment of leishmaniosis.61 It is a phospholipid analogue
that activates cellular proteases in Leishmania spp., which results
in apoptosis. Miltefosine is the first highly active oral drug for
treatment of leishmaniosis. Nevertheless, despite clinical improve-
ment in treated dogs, elimination of the parasite may not always
occur.62,63 Resistance to miltefosine can result from increased
P-glycoprotein–mediated drug efflux and decreased drug uptake.
Because miltefosine has a long half-life and must be administered
for 28 days, selection for resistant isolates has been a concern.
The World Health Organization has recommended that veteri-
nary use of miltefosine in dogs with leishmaniosis be avoided in
order to minimize the rate of selection for resistant strains.64
Over 90% of miltefosine is absorbed from the gastrointestinal
tract of dogs after oral administration.54 The half-life of miltefo-
sine in the dog is approximately 6 days, so it accumulates until a
steady-state plasma concentration is reached after 3 to 4 weeks
of treatment. Clearance is believed to result from slow hepatic
metabolism and excretion in bile. In dogs, adverse effects of treat-
ment are mild and transient and consist of occasional vomiting
and diarrhea.65 In contrast to meglumine antimoniate, miltefos-
ine does not appear to contribute to renal pathology in dogs.54,66
Antiprotozoal Drugs for Treatment
of Chagas’ Disease
Benznidazole
Benznidazole is a nitroimidazole that is used to treat acute Cha-
gas’ disease (see Chapter 78). Benznidazole is activated by a
parasite-specific nitroreductase to produce toxic metabolites.67
Resistance can result from reduction in the level of activity of
the nitroreductase and confers cross-resistance to nifurtimox.
Benznidazole is lipophilic, is readily absorbed from the gas-
trointestinal tract, and undergoes hepatic metabolism. In the
United States, it is available from the Centers for Disease Con-
trol and Prevention (CDC). The main adverse effect of treat-
ment in dogs is vomiting. In human patients, it can cause skin

rashes, peripheral neuropathy, and less commonly bone mar-
row suppression. It also has the potential to be carcinogenic.
Nifurtimox
Nifurtimox is a nitrofuran derivative used to treat Chagas’ dis-
ease in humans. It has limited efficacy, with 70% parasitologic
cure for acute disease and at best 20% for chronic disease.18
As with benznidazole, metabolic reduction of the nitro group
by a trypanosome-specific nitroreductase leads to the forma-
tion of toxic metabolites. In human patients, it is well absorbed
orally and undergoes hepatic metabolism. Gastrointestinal and
neurologic signs are the primary adverse effects of nifurtimox,
and like benznidazole, it has carcinogenic properties.68 Severe
adverse effects have precluded the use of nifurtimox in dogs.69
SUGGESTED READING
Rossignol JF. Cryptosporidium and Giardia: treatment options and
prospects for new drugs. Exp Parasitol. 2010;124(1):45-53.
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shedding and reshedding of Toxoplasma gondii oocysts in experi-
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23. Cohn LA, Birkenheuer AJ, Brunker JD, et  al. Efficacy of atova-
quone and azithromycin or imidocarb dipropionate in cats with
acute cytauxzoonosis. J Vet Intern Med. 2011;25:55-60.
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25. Pfefferkorn ER, Borotz SE, Nothnagel RF. Mutants of Toxoplasma
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26. Sykes JE, Dubey JP, Lindsay LL, et  al. Severe myositis associ-
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27. Vial HJ, Gorenflot A. Chemotherapy against babesiosis. Vet Para-
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28. Máthé A, Dobos-Kovács M, Vörös K. Histological and ultrastruc-
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30. Da Silva AS, Franca RT, Costa MM, et al. Experimental infection
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34. Solano-Gallego L, Baneth G. Babesiosis in dogs and cats—
expanding parasitological and clinical spectra. Vet Parasitol.
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elle of apicomplexan parasites as drug target. Curr Pharm Des.
2008;14:855-871.
36. Stephen B, Rommel M, Daugschies A, et al. Studies of resistance to
anticoccidials in Eimeria field isolates and pure Eimeria strains. Vet
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37. Altreuther G, Gasda N, Adler K, et  al. Field evaluations of the
­efficacy and safety of emodepside plus toltrazuril (Procox oral
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38. Altreuther G, Gasda N, Schroeder I, et  al. Efficacy of emodep-
side plus toltrazuril suspension (Procox oral suspension for dogs)
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1):S9-20.
39. Daugschies A, Mundt HC, Letkova V. Toltrazuril treatment of
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40. Lloyd S, Smith J. Activity of toltrazuril and diclazuril against Isos-
pora species in kittens and puppies. Vet Rec. 2001;148:509-511.
41. Petry G, Kruedewagen E, Bach T, et  al. Efficacy of Procox oral
suspension for dogs (0.1% emodepside and 2% toltrazuril) against
experimental nematode (Toxocara cati and Ancylostoma tubae-
forme) infections in cats. Parasitol Res. 2011;109(Suppl 1):S37-S43.
42. Voyvoda H, Pasa S, Uner A. Clinical Hepatozoon canis infection in
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45. Paila YD, Saha B, Chattopadhyay A. Amphotericin B inhibits entry
of Leishmania donovani into primary macrophages. Biochem Bio-
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46. Oliva G, Gradoni L, Ciaramella P, et  al. Activity of liposomal
amphotericin B (AmBisome) in dogs naturally infected with Leish-
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48. Solano-Gallego L, Koutinas A, Miro G, et  al. Directions for the
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51. Lima MI, Arruda VO, Alves EV, et  al. Genotoxic effects of the
antileishmanial drug Glucantime. Arch Toxicol. 2010;84:227-232.
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53. Ikeda-Garcia FA, Lopes RS, Ciarlini PC, et al. Evaluation of renal
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54. Bianciardi P, Brovida C, Valente M, et al. Administration of milt-
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2009;39:1055-1064:v-vi.

Basic Principles for Infection Control
Jane E. Sykes  and  J. Scott Weese
CHAPTER 11
Infection Control Programs for Dogs and Cats
Jane E. Sykes and J. Scott Weese
w KEY POINTS
• A  hospital infection control program consists of infectious
disease control personnel, a written protocol, training, and
documentation. The aim of such a program is to reduce the
incidence of hospital-acquired infections among patients, staff,
and visitors to a small animal hospital.
• The infection control program describes the requirement for
practices that optimize hygiene such as hand washing, the use
of protective clothing, cleaning and disinfection, and appropri-
ate disposal of infectious agents. The protocol can also be used
INTRODUCTION
The primary role of an infection control program is to reduce the
incidence of hospital-acquired infections (HAIs) by patients, staff,
and visitors to a small animal hospital. Infection control programs
in veterinary hospitals have largely evolved from evidence and
protocols from the human health care setting, together with our
understanding of transmission pathways for veterinary patho-
gens. In recent years, several factors have led to an increased rate
of adoption of infection control programs by veterinary hospitals,
such as an increased prevalence of multidrug-resistant bacterial
infections among small animal patients, the appearance of more
studies that support the common occurrence of transmission of
hospital-associated and zoonotic pathogens in the veterinary set-
ting, the requirement for an infection control program in veteri-
nary teaching hospitals for accreditation purposes, and increased
scrutiny of hospital-associated and zoonotic infections to protect
hospitals against litigation.
Infection Control Programs
Every small animal hospital should have an infection control
program. At a minimum, this should consist of an infectious
disease control officer, a written infection control protocol,
regular training of staff, and documents that record all training
SECTION 3
to educate staff about specific transmission precautions for
infectious diseases seen within a practice.
• This chapter describes the infection control program and
important components of an infection control protocol, includ-
ing the advantages and disadvantages of various methods of
sterilization, disinfection, and antisepsis used in small animal
hospitals.
• Information in this chapter also has relevance to infection con-
trol in shelters and in breeding and boarding facilities.
and surveillance efforts. In large hospitals, formation of an
infectious disease control committee may be required. In this
situation, the infectious disease control committee could con-
sist of an internist or criticalist with an interest or training in
infectious diseases, a veterinary clinical microbiologist, a nurs-
ing supervisor, a safety officer, the hospital administrator, and
the hospital director. There should be adequate personnel and
communication so that proper coverage is maintained when
one individual is absent or unavailable. All personnel who
work in a veterinary hospital, as well as visitors, should be
familiar with the infectious disease control personnel as well as
procedures and policies listed in the infection control protocol.
Documentation of training should be maintained for all hospi-
tal personnel.
The Hospital Infection Control Protocol
The objective of a hospital infection control protocol is to pro-
vide a standard procedure for the control of infectious diseases
in the hospital, in order to minimize animal-to-animal, animal-
to-human, and human-to-animal transmission of pathogens.
Adherence to the infection control protocol can also reduce
transmission of infectious agents between personnel through
increased hand washing and reduction of fomite contamination.
The infection control protocol is a legal document and should
105
106 SECTION 3  Basic Principles for Infection Control
be regularly updated by a designated hospital infection control
officer or committee. Each hospital should develop a protocol
that is tailored to address specific practice requirements and the
hospital design, purpose, and equipment used. Additional spe-
cial precautions may need to be described for hospitals that see
avian and exotic pet animal species, and in geographic locations
where serious zoonotic diseases such as rabies and plague are
endemic (see Chapters 13 and 55).
The infection control protocol details practices that optimize
hygiene such as hand washing, the use of protective clothing,
cleaning and disinfection, and appropriate disposal of infectious
agents. Specific infectious disease control procedures to be fol-
lowed in different areas of the hospital (radiology, surgery, the
intensive care unit, isolation, wards) can be included, as well
as policies on antimicrobial use. The protocol can also be used
to educate staff about routes of transmission, the potential for
zoonotic transmission, specific transmission precautions for
infectious diseases that are seen within the practice, and immu-
nization requirements, such as those for rabies (see Chapter 13).
Standard Precautions
All animals that enter a veterinary hospital are potential carri-
ers of pathogens that may be spread to other animals or people.
Animals may be colonized with multidrug-resistant bacteria,
often without showing evidence of disease due to these organ-
isms. Pet owners may carry these organisms on their hands and
clothing. Standard precautions such as hand hygiene and the
wearing of routine protective clothing can minimize the spread
of these bacteria around the hospital and to immunocompro-
mised animals.
Hand Hygiene
Frequent and proper hand and wrist washing to remove tran-
sient flora on the hands has been proven as the most important
component for prevention of the spread of infectious diseases
in human hospitals.1 Signs that outline proper hand-washing
technique, including the use of paper towels to turn off the fau-
cet, posted adjacent to basins around the hospital can improve
compliance among staff. Online videos that demonstrate proper
hand-washing technique are available for educational purposes.2
Guidelines for hand washing are shown in Box 11-1. Antibacte-
rial soap should be used, and all surfaces of the hands should be
rubbed together, which should include the backs of the hands,
between the fingers, and under the fingernails, for a total hand-
washing time of at least 20 seconds. In order to prevent chapped
skin, which can harbor bacteria, water used for hand washing
should not be too hot, and hand lotion should be applied regu-
larly. Behaviors such as keeping fingernails short, avoidance of
artificial and/or polished fingernails or hand jewelry, or wearing
jewelry on a chain around the neck instead of on the hand can
be encouraged. Meticulous hand hygiene is particularly impor-
tant for personnel who work frequently with immunocompro-
mised animals, such as emergency and critical care personnel.
The use of touch-free taps and paper towel dispensers can also
reduce transmission of bacteria during hand washing.
Alcohol-based hand sanitizers are a more convenient form of
sanitization. These can be provided in multiple locations around
a hospital, or travel-sized bottles can be carried in a coat pocket.
At least one to two full pumps or a 3-cm diameter pool of the
product should be dispensed onto one palm. All surfaces of the
hands and wrists should be rubbed with the product until it
BOX 11-1
Guidelines for Hand Washing and the Wearing
of Disposable Gloves
Situations That Require Hand Washing
Immediately before handling a patient
Immediately after handling a patient
Immediately before and after procedures that involve
nonintact skin
Before and after gloves are worn for procedures
After blood, body fluids, secretions, excretions, or con-
taminated items are touched
After cages are cleaned
Before and after eating, smoking, or leaving the hospital
After going to the restroom
Situations When Disposable Gloves Should
Be Worn
Contact with broken skin and bodily fluids (blood, urine,
and feces)
Handling of disinfectants
Handling of animals with suspected or known infectious
diseases
Handling of all animals for immunocompromised people
has dried. Use of soap and water, rather than hand sanitizer, is
recommended when there is gross contamination with organic
matter, or when exposure to alcohol-resistant pathogens such
as Clostridium spp. spores or parvovirus might have occurred.
However, the availability of hand sanitizers improves compli-
ance in busy hospital situations, is associated with lower rates of
dermatitis than medicated soaps, and is recommended for rou-
tine hand sanitation in human health care settings by the Cen-
ters for Disease Control and Prevention (CDC) and the World
Health Organization (WHO).3,4
The use of gloves prevents contamination of the hands with
microorganisms, prevents exposure to bloodborne pathogens,
and reduces the risk of transmission of microorganisms from
personnel to animals. However, gloves are not a substitute for
proper hand hygiene. Guidelines for wearing disposable gloves
are shown in Box 11-1. Gloves should be promptly removed
after use, before other surfaces are touched, and hands should
then be washed. If a glove is torn or punctured, it should be
removed and replaced as soon as possible.
Hospital Attire
Nonsurgical Areas
Staff should be encouraged to wear dedicated hospital attire
that is not worn elsewhere, so that hospital pathogens are not
transported to and from locations outside the hospital. At the
minimum, protective clothing such as clean laboratory coats or
hospital scrubs and closed-toe shoes must be worn in nonsurgi-
cal areas. Sleeves must be short enough or rolled up to expose
the wrists, and laboratory coats changed whenever gross soiling
occurs. In the absence of gross soiling, coats should be changed
daily. If neckties are worn, they must be secured in place by an
outer layer of clothing or a tie pin so that they cannot be con-
taminated as a result of contact with patients or environmental
 CHAPTER 11  Infection Control Programs for Dogs and Cats
TABLE 11-1
Transmission Precautions for Selected Infectious Diseases of Dogs and Cats
Infectious agent
Airborne Mycobacterium tuberculosis
Yersinia pestis
Francisella tularensis
Droplet Canine and feline transmissible respiratory disease pathogens
(e.g., canine distemper virus, canine respiratory coronavirus,
feline herpesvirus-1, feline calicivirus)
Contact Multidrug-resistant bacteria
Dermatophytes
Leptospira spp.
Salmonella spp.
Parvoviruses
surfaces and act as fomites, as has been shown to occur in
human hospital environments.5 Long hair must be tied back so
that it does not drape on animals and hospital surfaces. Face
shields and a clean gown should be worn during procedures
that are likely to generate splashes or sprays of blood and body
fluids. Gowns must be made of impervious material and tied on
securely and correctly. Soiled gowns must be removed as soon
as they are no longer required and face shields cleaned.
Surgical Areas
Dedicated operating room attire should be worn in surgical
areas and should be changed after gross soilage and when leav-
ing the operating room for the day. Caps and masks should
be worn and hands thoroughly washed on entry to the operat-
ing room. There is no evidence that dedicated footwear or foot
protection should be worn for prevention of surgical site infec-
tions in human patients, and major human guidelines do not
advocate that footwear be addressed.6 Protective wear should
be removed whenever staff members leave the operating room.
Traffic in and out of the operating room should be limited to the
minimum required for patient care.
General Animal Handling Precautions
All animals seen at a veterinary hospital should undergo a his-
tory and physical examination by a veterinarian to determine
the likelihood and nature of any transmissible infections that
might be present. Ideally, client beds, blankets, collars, and
leashes should not be brought into the hospital, where they
could become contaminated. Animals should always be placed
in cages that have been cleaned and disinfected appropriately.
Disposable thermometer sleeves should always be used on ther-
mometers. Equipment should not be shared between animals
unless it has been cleaned and disinfected. Diets that contain
raw meat and bones should not be fed or stored in the hospital,
because they commonly contain and can potentially transmit
foodborne gastrointestinal pathogens.7 The handling of sick
animals should be minimized, unless required for patient care.
Because some infections can be transmitted through bites
and scratches, staff should be educated on bite and scratch
avoidance, such as the use of restraint devices, protective gloves,
107
Specific precautions
Isolation, preferably in negative pressure facility
Properly fitted N95 respirator masks
Contact precautions
Isolation
Space animals at least 4 feet apart
Contact precautions
Warning signage
Gown, gloves, dedicated equipment
Isolation for select pathogens (see Box 11-2)
Limitation of movement
Standard hand hygiene precautions
Proper cleaning, disinfection, and disposal of
medical waste
and warning signage on cages and medical records. Should bites
or scratches occur, they should be vigorously flushed and imme-
diately washed with water and chlorhexidine or a dilute iodo-
phor solution, and the bite reported to appropriate officials as
necessary. Deep wounds could be irrigated with pressure using
a syringe without an attached needle. If a bite occurs, medi-
cal attention should be sought as soon as possible. All bites or
scratches must be documented and consideration be given as to
why the injury occurred, so that procedures or training to pre-
vent future injuries can be implemented if necessary.
Consumption of food and drink should be limited to parts of
a hospital where patient care and the handling of biologic speci-
mens and medications do not occur. Food and beverages should
not be left out open on benches for long periods. Microwaves
used for animal care purposes should not be used to heat food
intended for people.
Transmission-Based Precautions
Transmission-based precautions are instituted for selected
patients that are confirmed to be or suspected to be infected
or colonized with important transmissible pathogens. Trans-
mission-based precautions are used in combination with stan-
dard precautions. In the human hospital setting, three types
of transmission-based precautions have been developed—air-
borne, droplet, and contact precautions (Table 11-1).8 Airborne
precautions are used to prevent the transmission of diseases by
droplet nuclei (particles <5 µm). Transmission by droplet nuclei
occurs with diseases such as measles, varicella, and pulmonary
tuberculosis. The precautions for human patients involve isola-
tion in a single-bed, negative-pressure room and the wearing
of high-density respirator (N95) masks. These resemble surgi-
cal masks but filter 1-µm particles with an efficiency of at least
95% and must be properly fitted. Airborne contact precautions
are rarely necessary in hospitals that treat only dogs and cats,
but could be considered when animals suspected to have pneu-
monic plague, tularemia, or Mycobacterium tuberculosis infec-
tion. Droplet precautions are used to prevent transmission by
large-particle aerosols and do not require a negative-pressure
room. Droplet precautions apply to dogs and cats with trans-
missible respiratory disease. Contact precautions are indicated
108 SECTION 3  Basic Principles for Infection Control
for animals with infections that can be transmitted by direct
contact with the patient or through fomite contact.
If it is known in advance that an animal with a suspected
or known transmissible disease is to be seen at the hospital,
arrangements should be made to have the pet owner take the
animal from the parking area directly to an examination room,
so that the animal does not contaminate the waiting area. After
the animal has been examined, appropriate signage should be
placed on the door of the examination room to prevent use until
it has been properly cleaned and disinfected.
If hospitalization is required, animals with known or suspected
transmissible disease must be admitted either to an isolation ward
or to regular hospital cages with handling precautions, based on
the pathogen suspected. Immediately after hospitalization, the
name of the suspected or known pathogen should be posted on
the cage or run, together with a handling precautions notice. Ani-
mals that require contact precautions should be placed in cages
away from other animals in the ward and should not be moved
from one cage to another, unless there is a medical need. Animals
with transmissible respiratory disease should be placed in isola-
tion and separated both horizontally and vertically from other
patients by at least 4 feet. Gloves and a gown should be put on
before the patient is handled, and then removed and disposed of
immediately afterward. Hands should be washed after the gloves
are removed. Additional items of personal protective equip-
ment (masks, gowns, gloves, booties) may be required in some
circumstances. Soiled linen and equipment should be handled,
transported, and processed in a manner that prevents skin and
mucous membrane exposures and contamination of clothing and
that avoids transfer of microorganisms to other patients and envi-
ronments. All equipment that contacts the patient (scales, exami-
nation tables, stethoscope heads, floor) should be cleaned and
disinfected immediately after use. Medications and fluids from
these patients should not be returned to the hospital pharmacy.
When possible, personnel who handle these animals should not
work with other immunosuppressed animals in the hospital, or
they should work with the infectious disease cases last. If animals
with contact precautions must be moved within the hospital, per-
sonnel should ensure that precautions are maintained throughout
and that equipment and environmental surfaces that come into
contact with the patient are properly disinfected and/or disposed
of. The cleaning and bandaging of wounds infected with multi-
drug-resistant bacteria should be conducted in low-traffic areas
that can be properly cleaned and disinfected.
The owners of dogs and cats that have transmissible diseases
should be provided with general information regarding the risk
of disease transmission to in-contact animals and people that
includes the mode of transmission, duration of organism shed-
ding, and if there are special implications for young children or
other immunosuppressed individuals. If dogs and cats are diag-
nosed with a zoonotic disease, the owners should be notified
without delay. The owners should be told to see their physicians
if they become unwell or, in some circumstances, immediately,
and to advise a physician of the potential exposure.
Isolation
In contrast to human hospitals where patients can be more read-
ily isolated in single-bed rooms or cubicles, isolation of veteri-
nary patients can be more difficult because of the close proximity
of one animal to another. Floor contamination with secretions
and excretions can also occur more readily. Isolation rooms are
available in many veterinary hospitals, but they may be poorly
visible and/or accessible and may not provide access to an oxy-
gen source or be amenable to intensive monitoring and care. For
some animals (especially puppies and kittens) suspected to have a
transmissible disease, housing in a general ward or ICU area with
as much physical and procedural separation as possible, and with
strict infection control practices, may be acceptable (if perhaps
not optimal). Once a diagnosis is confirmed, the animal should
be moved immediately to isolation whenever possible. Patients
chosen for strict isolation vary based on the specific situation
and facilities available, but suggestions are provided in Box 11-2.
Only the individuals directly involved in the care of the patient
should enter isolation. Pet owners should not be allowed into
the isolation ward. No equipment used outside isolation (pens,
thermometers, stethoscopes, cell phones) should be brought into
isolation. Laboratory coats should be removed, and personnel
must put on protective wear such as a disposable gown, gloves,
and booties when entering the isolation ward. Face protection
may also be required, depending on the situation. A notice that
outlines the required precautions should be posted on the door
of isolation. Protective clothing should be removed before leav-
ing isolation, and hands should be washed. Once a patient has
been discharged, the room should be properly disinfected.
Handling of Potentially Infectious Materials
and Waste
The risk of human infection from patient blood and body fluids
in small animal hospitals is clearly lower than that in human
hospitals. However, a number of zoonotic pathogens have the
potential to be spread from dogs or cats to humans through
contact with medical waste, and a number of emerging zoonotic
infectious agents are bloodborne. Examples include Bartonella
spp., Anaplasma phagocytophilum, Brucella spp., and hemo-
plasmas. Proper handling of blood and body fluids also has the
potential to protect staff against as-yet-unrecognized zoonotic
pathogens. Improper disposal of medical waste in veterinary
hospitals risks injuring others who handle the waste and has the
potential to result in serious penalties or fines.
Potentially contaminated waste should be disposed of in an
approved plastic bag in a container labeled on all exposed sides
BOX 11-2
Examples of Infectious Agents of Dogs and Cats
for Which Strict Isolation Is Indicated
Salmonella spp.
Francisella tularensis
Yersinia pestis
Mycobacterium tuberculosis or Mycobacterium bovis
Microsporum canis
Rabies virus
Enteric viruses, such as parvoviruses
Canine transmissible respiratory disease pathogens
(Bordetella, canine distemper virus, influenza viruses,
canine respiratory coronavirus, canine adenovirus,
canine parainfluenza virus)
Feline upper respiratory tract disease pathogens (feline
herpesvirus-1, feline calicivirus, influenza virus,
Chlamydia)
 CHAPTER 11  Infection Control Programs for Dogs and Cats 109

with “biohazardous waste.” Blood-soaked materials, infected
materials, and empty fluid bags should all be placed in biohaz-
ardous waste containers. Care should be taken not to contami-
nate the outside of the container during disposal. The lid of the
biohazardous waste container must close properly. Blood and
body fluids should be inactivated with an appropriate disinfec-
tant (e.g., bleach or accelerated hydrogen peroxide) and allowed
to stand for 10 minutes before disposal. Disposal regulations
may vary depending on local laws, but liquid waste should not
be disposed of into storm drains.
Specimens for laboratory testing that are collected from a
patient with suspected zoonotic infectious diseases should be
placed in an outer plastic bag, taking care not to contaminate
the outside of the plastic bag, and labeled with an appropri-
ate warning label. Fecal material should be picked up using a
tongue depressor while wearing gloves, and placed in a sealed
plastic cup and clearly labeled. Urine specimens should be sub-
mitted in a sealed container.
Sharps Handling
Sharps handling practices receive considerable attention in human
medicine because of the risk of transmission of various blood-
borne pathogens. Although the risks are less in veterinary medi-
cine, significant injury or illness can follow sharps injuries, such
as transmission of infectious agents from the patient, allergic or
inflammatory reactions from exposure to medication, and inocu-
lation of opportunistic pathogens from the injured person’s own
skin microflora. Care should be taken to prevent injuries when
needles, scalpels, and other sharp instruments are used, cleaned,
or disposed of. Used needles should never be recapped, and they
should not be removed from the barrel of a disposable syringe.
Personnel should be instructed not to walk with uncapped nee-
dles and not to hold syringe or needle caps in the mouth. Used
needles should not be carried in a pocket. Animals suspected of
having an infectious disease that could be transmitted to humans
through a needle-stick injury should be sedated before skin
masses or peripheral lymph nodes are aspirated or venipuncture
is performed. Contaminated sharps (slides, scalpel blades, broken
glass, needles with attached syringes) must immediately be placed
into an approved puncture-resistant sharps disposal biohazard
container. Personnel who perform necropsy examinations should
take care to use sharp knives in the proper manner and to avoid
rushed situations. If a contaminated sharps injury occurs, medical
attention should be sought immediately if necessary.
Hospital Cleaning, Disinfection, and Sterilization
Definitions
Sterilization refers to complete elimination of all microbes,
including bacterial spores, and is accomplished in hospital set-
tings using processes such as pressurized steam, dry heat, ethyl-
ene oxide gas, or liquid chemicals.
Disinfection is the process that eliminates many or all
microbes from inanimate objects, but not bacterial spores. Fac-
tors that influence the efficacy of disinfection include the type of
microorganism present, their number, the amount and type of
organic matter present, the presence of biofilms, and the porosity
of the surface to be disinfected. Some disinfectants kill spores at
high concentrations and with prolonged exposure times. These
are known as chemical sterilants. At low concentrations and
short contact times, chemical sterilants inactivate all microbes
except large numbers of bacterial spores and are known as
high-level disinfectants. Low-level disinfectants inactivate most
vegetative bacteria, some fungi, and enveloped viruses, but
not bacterial spores. Intermediate-level disinfectants inactivate
mycobacteria, vegetative bacteria, most viruses, and most fungi
(Tables 11-2 and 11-3).
Antisepsis is the process that reduces the number of microbes
from living tissue and skin. Disinfectants are rarely used for skin
antisepsis because they can injure tissues and skin. Sanitation is
the reduction in the number of microorganisms on a surface to a
safe level. Germicides are agents that inactivate microorganisms
and include disinfectants, antiseptics, and sanitizers.

TABLE 11-2
Characteristics of Selected Disinfectants
Activity in the
Disinfectant ­Presence of
Category ­Organic Matter Advantages Disadvantages Precautions Comments
Alcohols: Rapidly Fast-acting Rapid evaporation Flammable Not appropriate for
Ethyl alcohol, inactivated No residue routine environmen-
isopropyl Relatively nontoxic tal dis­infection
alcohol Primarily used as
­antiseptics
Aldehydes: Good Broad spectrum Highly toxic Irritant Used as an aqueous
Formaldehyde, Relatively noncor- Carcinogenic solution or as a gas
glutaraldehyde rosive Requires ventilation (fumigation)
Alkalis: Unpleasant odor Do not mix with Not recommended for
Ammonia Irritating bleach. general use
Biguanides: Rapidly Nontoxic Incompatible with Not appropriate for
Chlorhexidine inactivated anionic detergents ­environmental dis­
infection
Primarily used as
­antiseptics
Continued
110 SECTION 3  Basic Principles for Infection Control

TABLE 11-2
Characteristics of Selected Disinfectants—cont’d

Activity in the
Disinfectant ­Presence of
Category ­Organic Matter Advantages Disadvantages Precautions Comments
Halogens: Rapidly Broad spectrum, Inactivated by Corrosive Used to disinfect clean
Hypochlorites inactivated sporicidal cationic soaps/ Irritant environmental
(bleach) Inexpensive detergents and May produce toxic ­surfaces
Can be used on sunlight. gas when mixed Only commonly
food preparation Frequent applica- with other chemi- available sporicidal
surfaces tion required. cals disinfectant
Oxidizing agents Good Broad spectrum Breakdown with Corrosive Excellent choice for
Environmentally time environmental
friendly ­disinfection
Phenolics Good Broad spectrum Toxic to cats Irritant Some residual activity
Noncorrosive Unpleasant odor after drying
Stable in storage Incompatible with
cationic or non-
ionic detergents
Quaternary Moderate Stable in storage Incompatible with Commonly used
­ammonium Nonirritating to skin anionic deter- ­primary environmen-
­compounds Low toxicity gents tal disinfectant
(QUATs) Can be used on food Some residual activity
preparation surfaces after drying
Effective at high tem-
peratures and pH

Source: Modified from the Canadian Committee on Antibiotic Resistance. Infection Prevention and Control Best Practices for Small Animal Veteri-
nary Clinics, 2008; http://www.wormsandgermsblog.com/2008/04/promo/services/infection-prevention-and-control-best-practices-for-small-animal-
veterinary-clinics/. Last accessed May 15, 2012.

TABLE 11-3
Antimicrobial Spectrum of Selected Disinfectants
Halogens: Quaternary
Alkalis: Biguanides: H­ ypochlorite Oxidizing Ammonium
Agent Alcohols Aldehydes Ammonia Chlorhexidine (Bleach) Agents Phenolics Compounds
Mycoplasmas ++ ++ ++ ++ ++ ++ ++ +
Gram-positive bacteria ++ ++ + ++ ++ ++ ++ ++
Gram-negative bacteria ++ ++ + + ++ ++ ++ +
Pseudomonads ++ ++ + ± ++ ++ ++ ±
Enveloped viruses + ++ + ++ ++ ++ ++ +
Non-enveloped viruses – + ± – ++ + ±* –
Fungal spores ± + + ± + ± + ±
Mycobacteria + ++ + – + ± ++ –
Bacterial spores – + ± – ++ + – –

++, Highly effective; +, Effective; ±, Limited activity; –, No activity.

*In general, phenols are not active against non-enveloped viruses but they do have some activity against rotaviruses. Activity against parvoviruses
has not been documented.
Examples of microorganisms from each category:
Mycoplasmas: Mycoplasma cynos, Mycoplasma felis; Gram-positive bacteria: Staphylococcus spp., Streptococcus spp.; Gram-negative bacteria: Bor-
detella bronchiseptica, Salmonella spp.; Pseudomonads: Pseudomonas aeruginosa; Enveloped viruses: influenza virus, herpesvirus; Non-enveloped
viruses: feline panleukopenia virus, canine parvovirus, feline calicivirus; Fungal spores: Aspergillus spp.; Acid-fast bacteria: Mycobacterium fortui-
tum; Bacterial spores: Clostridium difficile, Clostridium perfringens.
Source: Modified from the Canadian Committee on Antibiotic Resistance. Infection Prevention and Control Best Practices for Small Animal Veteri-
nary Clinics, 2008; http://www.wormsandgermsblog.com/2008/04/promo/services/infection-prevention-and-control-best-practices-for-small-animal-
veterinary-clinics/. Last accessed May 15, 2012.
 CHAPTER 11  Infection Control Programs for Dogs and Cats
TABLE 11-4
Recommended Conditions for Heat Sterilization
Conditions
Gravity displacement steam 121°C (250°F), 106 kPa (15
sterilization 132°C (270°F), 30 lb/in2
High-speed prevacuum steam 132°C (270°F), 30 lb/in2
sterilization
Dry heat sterilization 160°C (320°F)
170°C (340°F)
*Pressure settings may vary between incubators. When possible, follow manufacturer’s recommendations.
Cleaning involves the removal of all visible organic and
inorganic material from objects and surfaces through the use
of manual or mechanical processes and detergent or enzymatic
solutions.
According to the Spaulding method of classification, items
to be sterilized or disinfected can be grouped into critical, semi-
critical, and noncritical items.9 Critical items enter tissue or the
vascular system, or devices through which blood flows. Critical
items require sterilization before they can be used. Semicritical
items are items that contact mucous membranes or nonintact
skin and include endoscopes and balloon dilation catheters.
These generally require high-level disinfection. Noncritical
items are items that contact intact skin. Noncritical items gener-
ally require low-level or intermediate-level disinfection and con-
tact times of 1 to 10 minutes.9
All personnel should be educated regarding the standard
hospital germicides used for disinfection and antisepsis, how
they should be diluted and applied, and the hazards associated
with their use. Gloves and eye protection should be worn when
disinfectant solutions are handled or mixed. In large hospitals,
posters on hospital walls can be used to guide selection of appro-
priate germicides for different situations. Disinfectant solution
should be readily accessible throughout the hospital. Because
mops can spread infection, cotton mops should be laundered
daily, or mops with detachable microfiber heads should be used.
Microfiber heads absorb a large amount of water and are not
returned to the mop bucket after use. They can be laundered
and reused the following day.
Methods of Sterilization
Steam sterilization.  Steam sterilization involves the use
of saturated steam under pressure in an autoclave to achieve
sterilization. This is the most effective form of steriliza-
tion, is nontoxic and inexpensive, and as a result is the most
widely used as well. The use of steam under pressure allows
lower temperatures to be used for shorter periods of time
when compared with dry heat sterilization (Table 11-4). The
most common temperatures used for steam sterilization are
121°C (250°F) and 132°C (270°F). Cycle times vary depend-
ing on the autoclave used and whether items are wrapped or
unwrapped, but are generally less than 30 minutes (see Table
11-4). There are two basic types of autoclaves: gravity dis-
placement autoclaves, and high-speed prevacuum sterilizers.
Gravity displacement autoclaves admit steam at the top or
sides of the autoclave, which displaces air through a drain
vent at the bottom of the chamber (Figure 11-1). High-speed
111
Time
lb/in2)* 30 min wrapped items, 20 min unwrapped
15 min wrapped items
4 min
2 hours at temperature, 3 to 3.5 hours with cooling
1 hour at temperature, 2 to 2.5 hours with cooling
prevacuum sterilizers rapidly pump air out of the sterilizer
before steam is admitted. This leads to rapid penetration of
steam into all surfaces. As a result, cycle times can be reduced
to less than 15 minutes. Drying times are also reduced with
prevacuum sterilizers.
Sterilizers should be located away from potential sources of
contamination, such as sinks, trash disposal, or high-traffic areas.
Before steam sterilization is performed, instruments should be
cleaned thoroughly to remove organic and inorganic material,
and then dried. All jointed items should be opened or unlocked,
and items should not be crowded in the autoclave, so that steam
can circulate freely. At least 3 inches should be left between the
autoclave wall and items to be sterilized. The manufacturer’s
instructions for autoclave operation should be followed.
The efficacy of autoclaving must be tested for every auto-
claved item, with additional quality control performed on
a periodic basis. Autoclave indicator tape is routinely used;
although this only indicates conditions on the outside of the
package. Steam indicator strips should be included in each
surgical pack and evaluated by the person opening the pack.
Biological indicators provide a more definitive assessment of
autoclave efficacy and should be used periodically (e.g., weekly)
and the results documented. These consist of a standardized
population of bacterial spores, usually on a filter paper strip
or contained within a vial. The strip is then sent to the micro-
biology laboratory for culture, or cultured in-house, to ensure
that the spores have been completely inactivated by the steril-
ization process. Any indicator failure should result in immedi-
ate inspection of the autoclave. Sterilization indicators should
never be used as a substitute for proper autoclave operation
and careful preparation, packing, and loading of equipment to
be sterilized.
After steam sterilization, instruments should be allowed to
dry before they are removed, which typically takes an additional
30 minutes. Items should then be stored in a location and man-
ner that prevents further contamination.
Flash sterilization.  Flash sterilization refers to the rapid ster-
ilization of unwrapped instruments and is usually performed as
an emergency procedure in an operating room setting when time
is insufficient to perform the preferred sterilization of wrapped
items. In general it is performed for 3 minutes at 270°F and 27 to
28 lb/in2. Each instrument must be carefully protected to ensure
it does not become recontaminated during transport back to the
operating room, usually in a “flash pan.” Flash sterilization has
occasionally been associated with intraoperative infections and
should not be used for routine disinfection purposes.
112 SECTION 3  Basic Principles for Infection Control
FIGURE 11-1  Gravity displacement sterilization. Steam is admitted at the top or sides of the autoclave, which displaces air through a drain vent at the bottom of the chamber.
Gas sterilization.  Gas sterilization is used for sterilization
of heat- and moisture-sensitive instruments but can have sig-
nificant toxicity. Gases that can be used for sterilization include
formaldehyde, ethylene oxide (ETO), hydrogen peroxide vapor,
and ozone gases. The most commonly used gas in veterinary
medicine, ETO, has strong alkylating properties and causes pro-
tein coagulation, enzyme inactivation, and damage to nucleic
acid. When compared with heat sterilization, ETO sterilization
takes longer (24 hours or more including the time required to
allow ETO to diffuse out of packages at the end of sterilization)
and is more expensive. In addition, it only achieves surface ster-
ilization and requires sophisticated equipment and trained staff.
The gas is extremely flammable, irritates the eyes and mucous
membranes, is mutagenic and carcinogenic, and has a mislead-
ingly pleasant smell.
Irradiation.  Gamma irradiation uses a cobalt-60 radiation
source to destroy microorganisms through generation of high-
energy photons. Health care product manufacturers use gamma
irradiation to sterilize disposable medical supplies, such as cath-
eters, gloves, syringes, and pharmaceuticals.
Chemical sterilants.  Chemical sterilants are used when heat
or ETO gas sterilization is not available or would otherwise
damage instruments, such as endoscopes or laparoscopes. Dis-
infectants that act as sterilants when they are used at high con-
centrations and for adequate contact periods include certain
solutions that contain glutaraldehyde (e.g., >2.4% glutaral-
dehyde solutions, 0.95% glutaraldehyde with 1.64% phenol/
phenate), 0.55% ortho-phthalaldehyde (OPA), 7.5% hydrogen
peroxide, or greater than or equal to 0.2% peracetic acid (see
section on Disinfectants following). Chemical sterilants must be
rinsed repeatedly with sterile water and dried once sterilization
is complete. Chemical sterilants must be used with the proper
contact times and at the right concentration. Solutions may lose
efficacy, become diluted, or become contaminated with bacte-
ria over time. They should be replaced frequently according to
the manufacturer’s recommendations, and they should never be
used in procedures that involve sterile body sites unless there is
no other sterilizing option.
Disinfectants
Glutaraldehyde.  Glutaraldehyde is a saturated dialdehyde.
Glutaraldehyde solutions are relatively inexpensive, are non-
corrosive, and can be used to disinfect rubber, plastics, and
endoscopic equipment. Aqueous solutions require activation
by alkalinization to a pH of 7.5 to 8.5 for sporicidal activity
to occur. When alkalinized glutaraldehyde is used at a concen-
tration of 2.4% for adequate periods of time, either chemical
sterilization or high-level disinfection occurs, depending on the
contact time (e.g., Cidex). Contact times of at least 20 minutes
(at or above 20°C) are effective for high-level disinfection. Ster-
ilization requires a 10-hour contact time and higher concentra-
tions of glutaraldehyde (e.g., Cidex Plus, which contains 3.4%
glutaraldehyde), or formulations that combine glutaraldehyde
with another disinfectant.
Once activated, 2.4% glutaraldehyde solution retains activity
for 14 days, provided inadvertent dilution does not occur. The
solution is active in the presence of 2% organic matter. Inad-
vertent dilution can occur when endoscopes that contain fluid
within their channels are immersed in the solution. Test strips
are available from the manufacturer to monitor the activity of
the solution, but should not be used to extend the solution’s
expiration date. They indicate inactivity when the concentra-
tion drops below 1.5%, the minimum concentration required
for activity.
 CHAPTER 11  Infection Control Programs for Dogs and Cats
Glutaraldehyde irritates mucous membranes of the respi-
ratory and gastrointestinal tracts, and so endoscopes must be
rinsed properly after disinfection. It can also cause allergic
contact dermatitis, but it is not mutagenic or carcinogenic.
The wearing of nitrile rubber or butyl rubber gloves is recom-
mended. Because of its relative expense and toxicity, it is not
used to disinfect noncritical surfaces.
ortho-Phthalaldehyde.  OPA (e.g., Cidex OPA) is favored over
glutaraldehyde for high-level disinfection in the United States,
because it does not require activation, is stable over a wide pH
range, does not cause irritation of mucous membranes, and it
has a barely perceptible odor. Its activity is greater than that of
glutaraldehyde, and high-level disinfection is achieved with a
contact time of 12 minutes at or above 20°C.10,11 The primary
disadvantage of OPA is that it stains tissues and mucous mem-
branes gray. Protective equipment must be worn when handling
the solution, and it must be rinsed thoroughly from items after
treatment. Irritation can occur with eye contact. OPA is also
more expensive than glutaraldehyde.12 Solutions can be reused
for a maximum of 14 days.
Hydrogen peroxide.  Hydrogen peroxide (H2O2) is a potent
oxidizer. Hydrogen peroxide solutions are widely available, are
inexpensive, and can enhance removal of organic matter. When
used at concentrations of 7.5% for contact periods of at least
6 hours, hydrogen peroxide is a chemical sterilant (e.g., Spo-
rox). High-level disinfection can be achieved with contact times
of 12 to 30 minutes. Unfortunately 7.5% hydrogen peroxide
causes discoloration and functional changes within endoscopes
and so is not suitable for endoscope reprocessing. Although
nonirritating to mucous membranes, serious ocular damage can
occur with eye contact.
Accelerated hydrogen peroxide (AHP) is a patented hydrogen
peroxide solution that contains surfactants, an acid, and hydro-
gen peroxide. Use of a 4.5% AHP gel with contact times of 10
minutes can inactivate bacterial spores.13 Even 0.5% solutions
have some sporicidal activity and can inactivate non-enveloped
viruses such as canine parvovirus.14,15 AHP has gained popular-
ity as a disinfectant among health care institutions because it is
odorless, does not generate volatile gas, is nonirritating, and is
noncorrosive at dilutions used in health care settings.
Peracetic acid.  Peracetic acid belongs to the peroxygen fam-
ily of compounds. When used at 50°C to 56°C in a specific per-
acetic acid reprocessing system (Steris System 1, Steris), 0.2%
solutions achieve sterilization in very short time periods (30 to
45 minutes). Peracetic acid is active in the presence of organic
matter and may actually enhance its removal. Nevertheless
endoscopes must still be thoroughly cleaned before sterilization
to avoid fixation of blood onto the instrument. After steriliza-
tion, the processor rinses the instrument thoroughly. Peracetic
acid is stable but can be corrosive and causes discoloration of
endoscopes over time. It is more expensive than other chemi-
cal sterilants. Peracetic acid concentrates can cause irritation to
mucous membranes and are corrosive to the eye and skin, but
0.2% solutions are generally nonirritating.
Potassium peroxymonosulfate (Trifectant, Virkon S).  Like per-
acetic acid, potassium peroxymonosulfate is an oxidizing agent,
and 1% solutions are high-level disinfectants that are capable
of inactivation of non-enveloped viruses when contact times of
10 minutes are used.16 Thus it is suitable for inactivation of
canine parvovirus and feline calicivirus. Potassium peroxy-
monosulfate retains some activity in the presence of organic mat-
ter. Solutions are prepared from powder and remain active for
113
7 days. The powder is corrosive and can cause serious skin and
ocular burns, but the solution is nonirritating and less corrosive
than bleach. The solution stains fabric and may damage sur-
faces, particularly metal, over time if rinsing is not performed.
Sodium Hypochlorite (Bleach).  Household bleach, which con-
tains 5.25% to 6.15% sodium hypochlorite, is widely available,
inexpensive, and active in the presence of hard water. When
used at a 1:10 dilution for a 10-minute contact time, household
bleach is sporicidal but is irritating and can be highly corro-
sive to metal surfaces. This 1:10 dilution is used to control out-
breaks of clostridial diarrhea. For most hospital situations, 1:30
to 1:50 dilutions of household bleach provide more than 1000
ppm available chlorine and are effective for intermediate-level
disinfection. Noncritical surfaces can be disinfected with 1:500
dilutions of household bleach (>100 ppm available chlorine),
with contact times of at least 1 minute. Sodium hypochlorite
is inactivated by organic matter, so cleaning is required before
disinfection is performed. Solutions lose 50% of their activity
over a 1-month period unless they are stored in closed brown
bottles. Other disadvantages of bleach solutions are bleaching
of colored fabric and the release of toxic chlorine gas when
they are combined with other disinfectants such as quaternary
ammonium compounds (QUATs).
Quaternary Ammonium Compounds.  QUATs are often used
as low-level disinfectants for noncritical surfaces in health care
facilities. Although they are generally fungicidal, bactericidal,
and virucidal for enveloped viruses, some bacteria resist and
even grow within QUATs, and such contamination has resulted
in HAIs.17 Contact times vary by product so manufacturer rec-
ommendations should be followed; however, 10-minute contact
times are often used. Advantages of QUATs include their low
cost, high stability, and low toxicity. QUATs are inactivated by
hard water, organic materials, soaps, and detergents.
Phenolics.  Phenolics are one of the oldest known disinfec-
tant classes. Phenol derivatives that are available for hospital
disinfection include ortho-phenylphenol and ortho-benzyl-para-
chlorophenol (e.g., Qualitrol, Vetnex). Effective contact times
vary with the product. They are active against enveloped viruses
and bacteria, but they are not sporicidal and have limited activity
against fungi and non-enveloped viruses. Phenol derivatives are
active in the presence of organic matter and hard water, are stable,
and are noncorrosive. They irritate skin and mucous membranes,
and have the potential to be highly toxic if ingested by cats.
Because of these limitations and the availability of other effective
disinfectants, phenolics are rarely used in veterinary hospitals.
Antiseptics
Alcohol.  Ethyl alcohol and isopropyl alcohol are bactericidal,
virucidal (for enveloped viruses), and variably fungicidal. The
optimum bactericidal concentration is 60% to 90% in water
(volume/volume). They do not destroy bacterial spores or pen-
etrate proteinaceous material and so are not recommended for
high-level disinfection. They can be used to disinfect noncriti-
cal items such as hospital fomites and have very low toxicity,
so they are often included in waterless hand sanitizer products
together with emollients to prevent drying of the skin. They are
flammable and dry quickly, so it can be difficult to achieve ade-
quate contact times (≥1 minute). As a result, alcohol should not
be used for routine environmental disinfection.
Iodophors.  Iodine solutions are primarily used as skin anti-
septics. Formulations for disinfection are also available, which
contain higher concentrations of free iodine than antiseptic
114 SECTION 3  Basic Principles for Infection Control
preparations. An iodophor is a combination of iodine and a sol-
ubilizing agent (e.g., polyvinylpyrrolidone in povidone-iodine),
which serves to provide a sustained-release form of iodine.
Dilutions of iodophors are more active against microbes than
concentrated povidone-iodine, so iodophores must be diluted
correctly. Iodophors are bactericidal and virucidal. Their anti-
bacterial activity does not persist for long periods on skin or
in tissues, so frequent reapplication is required. Iodophors are
relatively nontoxic and nonirritating. They are stable in solu-
tion but are inactivated by organic material and they can stain
plastics and, to some extent, tissues.
Chlorhexidine.  Chlorhexidine is a cationic bisbiguanide that
disrupts microbial cell membranes and precipitates cell con-
tents. It is used widely for skin antisepsis in veterinary medicine.
Chlorhexidine has persistent activity on the skin, is nonirritat-
ing, is active in the presence of body fluids, and has rapid bacteri-
cidal activity. Like iodophors, chlorhexidine has limited activity
against fungi and mycobacteria. A 2% chlorhexidine solution is
preferred over 70% alcohol or povidone-iodine for skin prepa-
ration of central venous catheter sites in human patients18 and
is the skin antiseptic of choice for collection of blood for blood
cultures. Chlorhexidine may have slightly lower activity against
gram-negative bacteria and fungi than povidone-iodine, and
both chlorhexidine and iodophors have a slower antimicrobial
activity than 60% to 90% alcohol solutions.
Principles of Cleaning and Disinfection
Standard operating procedures that describe preparation and
application of disinfectants for all surfaces and objects used in
a veterinary hospital, as well as waste management, should be
developed, and staff and visitors should receive education about
the location of protocols and their use. Wards and procedure
and examination rooms should remain uncluttered in order to
facilitate effective cleaning. Because organic matter can inacti-
vate disinfectants, all visible debris should be removed before
disinfection. Disinfectants are only active when applied to clean,
nonporous surfaces. Porous surfaces such as dirt and wood can-
not be effectively disinfected using routine procedures. Hard
porous surfaces should be scrubbed with disinfectant using
brushes, and then rinsed with water after the contact time has
elapsed.
When hospital surfaces are cleaned, attention should be paid
to corners, under cabinets, chairs, the bases of examination
tables, door handles, elevator buttons, shelves, sinks, faucets,
and other surfaces that might otherwise be ignored. Personal
items such as cell phones and stethoscope heads should be regu-
larly disinfected with disinfectant wipes. Cell phones should be
disinfected at least daily, and stethoscope heads wiped between
patients. Stethoscope tubing should be cleaned regularly with
soap and water. Disinfection of other fomites should be per-
formed on a regular (at least daily) basis. These include digital
thermometers, computer keyboards and mice, land-line tele-
phones, calculators, microscopes, otoscopes, and blood pres-
sure cuffs.
Cleaning staff should wear gloves when general hospital
disinfection is performed. This may not be necessary for spot
cleaning of areas such as examination room tables or fomites.
Depending on the disinfectant, additional protective attire, such
as a gown, boots, or face mask, may be required if there is a
probability of significant splashing during the disinfection pro-
cess. After disinfection is complete, protective clothing should
be removed and handwashing performed. The individuals
responsible for cleaning and disinfection at each time point, sit-
uation, and location in the hospital should be clearly identified.
A plan for the cleaning of outdoor areas that become contami-
nated with excretions or other biohazardous material should be
outlined.
When disinfection of cages or runs is performed, animals
should be removed and placed in a clean holding cage or run,
away from other patients. Solid waste and soiled laundry or
paper should be removed and placed directly in waste or laun-
dry containers, taking care to avoid dripping onto the floor. If
laundry is contaminated with potentially infectious material, it
must be bagged and labeled with the contents and suspected
infectious agent. Proper contact times should be used after
application of disinfectant solution. Runs and cages should be
allowed to dry completely before a new patient is introduced.
Immunocompromised People and Children
People are considered immunocompromised if they (1) have
various comorbidities such as diabetes mellitus, chronic kidney
failure, leukopenia, immune-mediated disease, HIV/AIDS, con-
genital immunodeficiencies, hepatic cirrhosis, cancer, or sple-
nectomy; (2) are being treated with immunosuppressive drugs
or chemotherapeutics; (3) are very young (5 and under) or
very old; or (4) are pregnant, although the immunosuppressive
effects of pregnancy are considerably lower than those of the
preceding disorders.
Immunocompromised staff members who work in small
animal hospitals should discuss any necessary work restrictions
and precautions in light of their specific condition with their
physician or an infectious disease doctor. The risks for a dia-
betic, for example, may be significantly different from those for
a transplant recipient. In general, immunosuppressed individu-
als who work in small animal hospitals should avoid handling
patients with suspected or known infectious diseases. Gloves
should always be worn when handling animals and animal
fluids or excreta, and strict attention to general hand hygiene
and any bites, scratches, or sharps injuries is critical. Children 5
years of age or younger should be kept out of patient care areas.
Strict attention to hygiene and protective apparel is necessary
when handling soiled cat litter or animal feces, because of the
risk for transmission of enteropathogens. The reader is referred
to other chapters in this book for public health implications of
specific infectious diseases that may be encountered in small ani-
mal hospitals. If possible, small animal clinics should establish
a relationship with a doctor who is prepared to consult on zoo-
notic exposures with staff members.
Transmissible Disease Surveillance
and Reporting
A transmissible disease surveillance program allows collection
of baseline data that establishes the prevalence of certain infec-
tious diseases, so that possible outbreak situations can be readily
identified. It can also provide background prevalence informa-
tion on antimicrobial drug susceptibility patterns, which can
assist in the initial selection of antibacterial drugs for individual
animal patients while culture and susceptibility test results are
in progress. Information is generally collected for multidrug-
resistant bacterial infections, zoonotic diseases, highly conta-
gious diseases, pathogens that are difficult to inactivate with
 CHAPTER 11  Infection Control Programs for Dogs and Cats
disinfectants, or agents of regulatory concern. Although surveil-
lance may seem like a difficult, time-consuming, and expensive
measure, in reality, it can be easy and cost-effective and can
provide important information.
There are two main forms of infection control surveillance
applicable to veterinary hospitals: active and passive. Active
surveillance involves collection of data specifically for infection
control purposes. This can provide the highest quality and most
relevant information, but it can be expensive and time consum-
ing. Examples of this would be collection of swab specimens
to screen for infection with methicillin-resistant Staphylococ-
cus pseudintermedius (MRSP) from dogs before surgery as part
of MRSP outbreak investigation. Active surveillance is a core
component of infection control in most human hospitals, but
it is only sporadically used in veterinary medicine and is rarely
needed in most veterinary clinics. It is typically reserved for
large facilities with increased infection control risks and per-
sonnel available to direct such testing, or as a part of outbreak
control.
In contrast, passive surveillance is a practical, easy, and
cost-effective surveillance approach that can and should be per-
formed in every veterinary clinic. It involves the use of data that
are already available, such as information about surgical site
infections collected during routine follow-up or culture results
from clinical testing. The quality of passive surveillance data
can be limited by poor or incomplete record keeping or sporadic
use of appropriate diagnostic tests, but if properly collected and
if potential biases are understood, passive surveillance data can
provide important insight into aspects such as endemic disease
rates, common pathogens, and antimicrobial susceptibility
trends. To facilitate passive surveillance, clinicians should be
encouraged to use appropriate diagnostic testing to determine
the etiology of nosocomial infections, even if the clinical con-
sequences are not severe. They also should be encouraged to
confirm a diagnosis in animals with suspected transmissible dis-
ease. This allows clients to protect their other animals and their
families and friends who might be in contact with the pet, and
it benefits the hospital.
Another form of surveillance that is easy to perform and
potentially very useful is syndromic surveillance. This involves
surveillance for readily identifiable syndromes (i.e., diarrhea,
cough) instead of specific diagnoses. Although syndromes do not
indicate a specific disease, they can indicate an increased risk of
infectious disease. Syndromic surveillance is an initial screening
tool that can be used by all personnel, including lay personnel,
to flag potentially high-risk cases and allow for early implemen-
tation of enhanced infection control practices. All clinic person-
nel should be made aware of certain syndromes that indicate
the need either for isolation or for further investigation, such
as diarrhea, fever of unknown origin, acute neurologic disease,
wound infections, and acute respiratory tract disease. Protocols
to deal with these animals on arrival should be developed. As
an example, a dog with an acute onset of cough should be con-
sidered potentially infectious, and if front office personnel note
this syndrome at the time the appointment is made, the dog
can be handled appropriately on arrival (e.g., admitted directly
to isolation or an examination room, with personnel wearing
enhanced barrier precautions from the onset). The role of lay
staff (i.e., front office stall) is critical, as these people are the
ones who are most able to identify such cases before they enter
the clinic and ensure that they are properly handled to prevent
nosocomial or zoonotic transmission.
115
BOX 11-3
Infectious Diseases of Dogs and Cats That Are Reportable or
Potentially Reportable to Public Health Agencies
Amebiasis Q fever
Granulocytic anaplasmosis Rabies
Brucellosis Salmonellosis
Campylobacteriosis Tularemia
Coccidioidomycosis Tuberculosis
Cryptococcus gattii MRSA (not MRSP)
infection Novel H1N1 influenza
Cryptosporidiosis virus infections
Giardiasis West Nile Virus infection
Leishmaniosis Yersinia infections
Leptospirosis
Lyme disease
Environmental cultures are rarely informative and so are not
considered a useful routine infection control tool. They may
be used to detect a specific pathogen if an outbreak of an HAI
is suspected when there is suspicion that the environment is a
source of exposure. However, it is often difficult to distinguish
cause from effect (i.e., environmental contamination that leads
to transmission vs. environmental contamination that occurs as
a result of contamination from a patient in the absence of a risk
of transmission).
Although surveillance programs require time, effort, and
expense, in the long term they may save morbidity and mortal-
ity and reduce costs that relate to control of large outbreak situ-
ations or the legal ramifications of HAIs.
When zoonotic diseases are identified, all in-contact individ-
uals and, if necessary, public health authorities should be noti-
fied in the appropriate manner. The specific diseases that must
be reported to authorities vary among geographic locations.
Examples of diseases that may be of interest to public health
authorities are listed in Box 11-3.
Surgical Preparation
Surgical site infections (SSIs) are an uncommon but important
and sometimes devastating complication of surgery. Every
patient undergoing a surgical procedure is at some risk of SSI.
Standard practices have been developed to reduce the risk of
SSIs. Although a wide range of practices are relevant, prepara-
tion of the patient and preparation of the surgeon are critical.
Patient Preparation
The patient’s endogenous microflora is an important source of
pathogens that cause SSIs. Careful preparation of the patient,
therefore, can help reduce contamination of the surgical site
during surgery. The goal of preoperative surgical site manage-
ment is to eliminate potential pathogens while not creating a
physical environment that is more conducive to bacterial colo-
nization or infection postoperatively. Bathing of the patient pre-
operatively is reasonable if there is significant contamination of
the haircoat19 and if the patient’s coat can be dried by the time
of surgery. In most situations, bathing is not required. Rather,
careful hair removal and skin antisepsis are the main measures.
116 SECTION 3  Basic Principles for Infection Control
The goals of surgical scrubbing of patients are to reduce bacte-
rial counts, reduce debris, and facilitate later antisepsis. Scrub-
bing should be done as atraumatically as possible. Minimizing
skin damage during clipping and scrubbing is essential, because
skin damage from excessive attempts to clip all remaining
pieces of hair or from forceful scrubbing of the surgical site
can create an environment that is more amenable to bacterial
growth. This predisposes to infection rather than reducing the
risk. Clipping should be performed after anesthesia, to reduce
the risk of trauma associated with a struggling patient and
to minimize the time between clipping and surgery. Clipping
should be done outside the operating environment. There is cur-
rently no information that relates to optimal methods of clean-
ing and disinfecting clippers. Repeated use of clipper blades
without sterilization not surprisingly results in higher levels of
bacterial contamination of blades20; however, the clinical rel-
evance of this is unclear, because the surgical site is cleaned
and disinfected after clipping. Regular cleaning and disinfection
of clippers are probably useful, and they should be thoroughly
cleaned and disinfected after use on an animal with a poten-
tially transmissible infection (e.g., an animal with diarrhea), on
an area where the skin is broken, or on any area where the skin
or hair is significantly contaminated with feces, urine, blood, or
other body fluids.
After hair removal, various approaches for skin antisepsis
can be used. There is little outcome-based evidence of the rela-
tive efficacy of different approaches in the human literature.
Typically, a three-step process is used, with initial scrubbing
of the site with biocidal (i.e., chlorhexidine, povidone-iodine)
soap, followed by application of alcohol for biocidal effects and
to remove oils, and a final application of a biocidal solution
with residual activity (e.g., chlorhexidine).
Surgeon Preparation
The surgeon’s body and clothing are potential sources of SSI
pathogens, and standard practices have been developed to
reduce the risk of contamination of the surgical site. One aspect
is the use of proper protective clothing. Every person in the
operating environment should wear clean surgical scrubs. These
scrubs should be dedicated for use only in the operating room
or should be covered with a clean laboratory coat whenever the
individual is outside of the operating room.
Surgical hand antisepsis is critical because of the close con-
tact of the hands with the surgical site and the relatively high
incidence of grossly evident breaks or micro-breaks in gloves.
Surgical hand antisepsis is designed to greatly reduce bacte-
rial burdens on the hands, particularly the abundant transient
microflora that contains most of the relevant pathogens that
cause SSIs. Surgical antisepsis must find a balance between
effective elimination of pathogens, minimization of trauma to
the skin (because skin irritation facilitates bacterial growth),
and time constraints. The traditional approach to surgical hand
antisepsis has involved structured scrubbing of the hands for a
predetermined time. Recommended scrub times vary between
products but are typically 2 to 4 minutes.
Application of alcohol-chlorhexidine combinations has been
evaluated as a replacement for surgical scrubbing and has been
shown to be more effective than standard surgical scrub meth-
ods.21,22 Specific manufacturer instructions should be followed,
and hands and arms must be dry before application of gloves.
The use of alcohol-based surgical hand antisepsis products is
encouraged as a replacement for traditional scrubbing.
BOX 11-4
Example of Criteria Specified within a Hospital
for Use of Restricted Antimicrobial Drugs
• Infection must be documented based on clinical abnor-
malities and culture.
• Subclinical infections should not be treated with these
antibiotics. These antibiotics should be reserved for
infections that are life threatening.
• Resistance to all other reasonable options and suscepti-
bility to the chosen antimicrobial must be documented.
• The infection must be potentially treatable.
• The clinician should contact the infectious disease
control officer or a clinical microbiology faculty
member by email or telephone to discuss antimicrobial
susceptibility test results, determine whether there are
any other viable options, and confirm that a restricted
antimicrobial is necessary.
Regardless of the method used, a thorough handwash with
careful cleaning under the fingernails must be performed at the
beginning of each day.23 Long (>1⁄4-inch) and artificial nails are
prohibited in many human health care facilities and some vet-
erinary hospitals because they harbor pathogenic bacteria24 and
are associated with surgical glove tears.
A cap and mask must be worn during hand antisepsis, and
sterile gown and gloves must then be donned using appropriate
technique.
Antimicrobial Drug Use
The use of antimicrobial drugs contributes to the selection for
antimicrobial-resistant bacteria. Inclusion of a policy on anti-
microbial drug use in the infection control protocol may help
to reduce the prevalence of multidrug-resistant HAIs. On their
own, fever and leukocytosis are not justification for antimicro-
bial drug treatment. Whenever possible, when infection is sus-
pected, attempts to obtain material for culture and susceptibility
testing should be made before antimicrobial drug treatment is
initiated. When secondary bacterial infection is likely, attempts
to identify and treat the underlying cause should be made. In
an effort to minimize the emergence of resistance in bacterial
flora of animals in the hospital and decrease the risk of resis-
tant HAIs, the use of several antimicrobials (e.g., certain par-
enteral third-generation cephalosporins, vancomycin, linezolid,
and carbapenems such as imipenem and meropenem) may be
restricted unless criteria must be met (Box 11-4).
Prevention of Infectious Disease in Shelters
and Breeding and Boarding Facilities
Attention to hand hygiene and proper disinfection of fomites
and the environment as outlined for hospitals can also be used
to reduce transmission of infectious pathogens in shelters and
in breeding and boarding facilities (Box 11-5). Disinfectants
with activity against parvoviruses and feline calicivirus should
be used routinely, such as a 1:30 dilution of household bleach,
 CHAPTER 11  Infection Control Programs for Dogs and Cats
BOX 11-5
Factors That Should Be Considered for the Reduction of
Infectious Disease Transmission in Animal Housing Facilities
Adequate ventilation and air quality
Adequate temperature
Adequate lighting conditions (including provision of dark-
ness at night)
Population density/space per animal
Daily removal of fecal and urine contamination and
disinfection
Adequate drainage
Separate housing areas for dogs and cats
Isolation for sick animals
Separate housing areas for young and adult animals
Individual housing or small groups of two to four compat-
ible animals
Separation of elimination, feeding, and resting areas
Elevated resting areas
Elevated cages for cats
Provision of hiding areas
Sound control
Use of surfaces that can be readily disinfected
Proper use of appropriate disinfectants
Hand hygiene and protective clothing
Fomite control
Use of appropriate vaccination protocols
Order of care (young animals, healthy adults, and then
sick animals)
Proper nutrition
Pain management
Free access to clean water
Rodent and pest control
Parasite control
Daily monitoring by properly trained individuals
Proper diagnosis of disease outbreaks
potassium peroxymonosulfate, or accelerated hydrogen perox-
ide solutions. Removal of organic matter, the use of surfaces
that can be adequately disinfected, and proper contact times are
essential.
Factors that increase stress should also be minimized, such
as high population densities, the grouping of animals, and poor
nutrition. Cats and dogs should be separated from one another.
In cats, the use of methods that reduce stress, such as provision
of a low-stress cage environment, can dramatically reduce the
frequency of upper respiratory tract disease. Compartmental-
ized housing allows animals to urinate and defecate away from
areas where they rest and eat, and provision of a hiding area so
that animals can retreat from visual stimulation can also reduce
stress. A total of 10 to 20 air changes per hour is often rec-
ommended for animal housing, but higher levels of ventilation
may be needed with increased population density and concen-
tration of airborne contaminants. Cages should be cleaned and
disinfected at least daily. The use of compartmentalized housing
areas also facilitates cleaning and disinfection without requir-
ing an animal to be removed from the housing area. Isolation
areas for sick animals should be present and these should have
117
separate airflow from areas that house healthy animals. Mass
treatment of dogs and cats with upper respiratory tract disease
with antimicrobials without attention to the underlying causes
only results in selection for antimicrobial drug resistant organ-
isms and is not recommended. Detailed information on the con-
trol of infectious diseases in shelter environments is beyond the
scope of this book but can be found elsewhere.25 The American
Association of Shelter Veterinarians and the Humane Society
of the United States have published guidelines for operation of
animal shelters.26,27
SUGGESTED READINGS
Association of Shelter Veterinarians. Guidelines for standards of care
in animal shelters. 2010. oacu.od.nih.gov/disaster/ShelterGuide.pdf.
Last accessed November 17, 2012.
Boyce JM, Pittet D. Guideline for hand hygiene in health-care set-
tings. MMWR. 2002;51. http://www.cdc.gov/handhygiene/Guideli
nes.html. Last Accessed November 17, 2012.
Canadian Committee on Antibiotic Resistance. Infection
prevention and control best practices for small animal veterinary
clinics. 2008. http://www.wormsandgermsblog.com/2008/04/promo/
services/infection-prevention-and-control-best-practices-for-small-
animal-veterinary-clinics/. Last accessed November 17, 2012.
WHO guidelines on hand hygiene in health care. http://www.cdc.gov/
handhygiene/guidelines.html. Last accessed November 17, 2012.
REFERENCES
1. Edmond EB, Wenzel RP. Isolation. In: Mandell GL, Bennett JE,
Dolin R, eds. Principles and Practice of Infectious Diseases. 7th ed.
Philadephia, PA: Elsevier; 2010:3673-3676.
2. Longtin Y, Sax H, Allegranzi B, et al. Hand hygiene. N Engl J Med.
2011:e24.
3. WHO guidelines on hand hygiene in health care. 2009. http://
www.cdc.gov/handhygiene/Guidelines.html. Last accessed Novem-
ber 17, 2012.
4. Guideline for hand hygiene in health-care settings. 2002. http://
www.cdc.gov/handhygiene/Guidelines.html. Last accessed Novem-
ber 17, 2012.
5. McGovern B, Doyle E, Fenelon LE, et al. The necktie as a potential
vector of infection: are doctors happy to do without? J Hosp Infect.
2010;75:138-139.
6. Centers for Disease Control and Prevention. Guideline for the pre-
vention of surgical site infection. 1999. http://www.cdc.gov/HAI/
ssi/ssi.html. Last accessed November 17, 2012.
7. Lenz J, Joffe D, Kauffman M, et al. Perceptions, practices, and con-
sequences associated with foodborne pathogens and the feeding of
raw meat to dogs. Can Vet J. 2009;50:637-643.
8. Guideline for isolation precautions: preventing transmission of
infectious agents in healthcare settings. 2008. http://www.cdc.gov/
hicpac/2007ip/2007isolationprecautions.html. Last accessed Novem-
ber 17, 2012.
9. Rutala WA, Weber DJ. Disinfection, sterilization, and control of
hospital waste. In: Mandell GL, Bennett JE, Dolin R, eds. Principles
and Practice of Infectious Diseases. 7th ed. Philadephia, PA: Elsevier;
2010:3677-3695.
10. Akamatsu T, Minemoto M, Uyeda M. Evaluation of the antimicro-
bial activity and materials compatibility of orthophthalaldehyde as
a high-level disinfectant. J Int Med Res. 2005;33:178-187.
11. Hession SM. Endoscope disinfection by ortho-phthalaldehyde
in a clinical setting: an evaluation of reprocessing time and costs
compared with glutaraldehyde. Gastroenterol Nurs. 2003;26:
110-114.
12. Cooke RP, Goddard SV, Whymant-Morris A, et al. An evaluation
of Cidex OPA (0.55% ortho-phthalaldehyde) as an alternative
to 2% glutaraldehyde for high-level disinfection of endoscopes.
J Hosp Infect. 2003;54:226-231.
118 SECTION 3  Basic Principles for Infection Control
13. Omidbakhsh N. Evaluation of sporicidal activities of selected
environmental surface disinfectants: carrier tests with the spores
of Clostridium difficile and its surrogates. Am J Infect Control.
2010;38:718-722.
14. Alfa MJ, Lo E, Wald A, et al. Improved eradication of Clostridium
difficile spores from toilets of hospitalized patients using an acceler-
ated hydrogen peroxide as the cleaning agent. BMC Infect Dis. 2010;
10:268.
15. Howie R, Alfa MJ, Coombs K. Survival of enveloped and non-
enveloped viruses on surfaces compared with other micro-organ-
isms and impact of suboptimal disinfectant exposure. J Hosp
Infect. 2008;69:368-376.
16. Eleraky NZ, Potgieter LN, Kennedy MA. Virucidal efficacy of four
new disinfectants. J Am Anim Hosp Assoc. 2002;38:231-234.
17. Weber DJ, Rutala WA, Sickbert-Bennett EE. Outbreaks associated
with contaminated antiseptics and disinfectants. Antimicrob Agents
Chemother. 2007;51:4217-4224.
18. Maki DG, Ringer M, Alvarado CJ. Prospective randomised trial
of povidone-iodine, alcohol, and chlorhexidine for prevention of
infection associated with central venous and arterial catheters. Lan-
cet. 1991;338:339-343.
19. Stick JA. Preparation of the surgical patient, the surgery facility,
and the operating team. In: Auer JA, Stick JA, eds. Equine Surgery.
3rd ed. Philadelphia, PA: Saunders Elsevier; 2006:123-140.
20. Masterson TM, Rodeheaver GT, Morgan RF, et al. Bacteriologic
evaluation of electric clippers for surgical hair removal. Am J Surg.
1984;148:301-302.
21. Mulberrry G, Snyder AT, Heilman J, et al. Evaluation of a water-
less, scrubless chlorhexidine gluconate/ethanol surgical scrub for
antimicrobial efficacy. Am J Infect Control. 2001;29:377-382.
22. Hobson DW, Woller W, Anderson L, et  al. Development and
evaluation of a new alcohol-based surgical hand scrub formulation
with persistent antimicrobial characteristics and brushless applica-
tion. Am J Infect Control. 1998;26:507-512.
23. Larson EL. APIC guideline for handwashing and hand antisepsis in
health care settings. Am J Infect Control. 1995;23:251-269.
24. Pottinger J, Burns S, Manske C. Bacterial carriage by artificial ver-
sus natural nails. Am J Infect Control. 1989;17:340-344.
25. Miller L, Hurley KF. Infectious Disease Management in Animal
Shelters. Ames, IA: Wiley-Blackwell; 2009.
26. Humane Society of the United States. HSUS guidelines for stan-
dard operating procedures for animal shelters. http://dev.animal
sheltering.pub30.convio.net/resource_library/policies_and_
guidelines/guidelines_for_standard_operating_procedures.html.
Last accessed November 17, 2012.
27. Association of Shelter Veterinarians. Guidelines for standards
of care in animal shelters. 2010. oacu.od.nih.gov/disaster/Shelter
Guide.pdf. Last accessed November 17, 2012.
CHAPTER 12
Immunization
Jane E. Sykes
w KEY POINTS
• A  ctive immunization can partially or completely protect dogs
and cats from severe consequences of infection with a variety
of different pathogens, and in some cases it reduces shedding
of these pathogens.
• Vaccines contain attenuated live microorganisms, inactivated
microorganisms, or portions of these organisms. They also con-
tain preservatives and adjuvants.
• Failure of immunization can occur with improper storage or
administration of vaccines, a large challenge dose, host factors
such as concurrent infections or disease, and interference by
maternal antibody.
INTRODUCTION
Immunization refers to artificial induction of immunity or protec-
tion from infectious disease and may be active or passive. Active
immunization involves administration of vaccines that stimulate
cell-mediated or humoral immunity, or both, to a specific patho-
gen. Passive immunization refers to the administration of antibod-
ies in order to provide temporary protection from disease and can
occur through acquisition of maternally derived antibody (MDA)
transplacentally, in colostrum, or milk; or treatment with prepara-
tions that contain specific or nonspecific immunoglobulins (see
Immunomodulators, Chapter 7, and post-exposure prophylaxis for
rabies, Chapter 13). Readers are referred to advanced immunology
texts for detailed descriptions of the physiology of active and pas-
sive immunity.1
The goal of immunization is to generate a protective immune
response of prolonged duration against a specific infectious dis-
ease, with minimal adverse effects. Because of the potential for
adverse effects, vaccination should be performed only if there
is a risk for significant morbidity or mortality from an infec-
tious disease. Since the 1950s, a huge number of vaccines for
dogs and cats have been developed and marketed worldwide,
and more are in development. Nevertheless, it is estimated that
even in developed countries such as the United States, only 30%
to 50% of dogs are properly immunized, and possibly an even
smaller proportion of cats.2,3 Appropriate vaccination of a larger
proportion of the pet population may assist in reduction of the
prevalence of infectious diseases through the induction of herd
immunity.
With the appearance of injection-site sarcomas in cats, increased
emphasis has been placed on vaccine safety, and a change from
annual to 3-yearly immunization protocols for some vaccines has
been recommended, with administration of other vaccines based
on exposure risk. Vaccines have had a profound influence in the
control of infectious disease, and for many vaccines the benefits of
vaccination outweigh the risks.
• O  ther adverse effects of vaccine administration are uncommon
to rare but include hypersensitivity reactions, disease induced
by live attenuated vaccine organisms, and injection-site sarco-
mas in cats.
• The decision to administer a vaccine should be based on dis-
cussion of risks and benefits between the veterinarian and pet
owner. This should be documented in the medical record.
• Guidelines for vaccine selection and administration have been
published by a number of veterinary bodies, such as the AAFP,
AAHA, AVMA, and WSAVA; suggestions can also be found in
Appendix I.
Vaccine Composition and Types of Vaccines
A vaccine is a suspension of attenuated live or inactivated
microorganisms, or parts thereof, that is administered to induce
immunity. In addition to protective antigens, vaccines may
contain preservatives and stabilizers as well as specific antibi-
otics to preserve the antigen and inhibit bacterial and fungal
growth within the vaccine. Some vaccines also contain an adju-
vant to enhance the immune response to the antigen. Although
the mechanisms are not completely clear, adjuvants can delay
the release of antigen from the site of injection and induce the
secretion of chemokines by leukocytes.4 The most widely used
adjuvants are particulate adjuvants, such as those that contain
aluminum salt precipitates such as aluminum hydroxide.5 Other
particulate adjuvants include immunostimulators such as sapo-
nin, which is present in a canine Leishmania vaccine.
Attenuated live vaccines (or modified live vaccines) con-
tain microorganisms that are artificially manipulated so as to
negate or greatly reduce their virulence, or are field strains of
low virulence. Repeated passage through cell culture is the most
common means of attenuation. Because they replicate in the
host, organisms in attenuated live vaccines usually stimulate an
immune response that most closely mimics the protection that
results from natural infection. Vaccination with attenuated live
canine parvovirus (CPV) and canine distemper virus (CDV) vac-
cines in the absence of MDA can result in protective immune
responses within 3 days of a single injection, which may be
followed by immunity that lasts many years, if not for life.6-8
Partial immunity after vaccination with attenuated live CDV
and feline panleukopenia virus (FPV) vaccines can occur within
hours.3,9,10 In addition, vaccine organisms that are shed can
serve to immunize other animals in a population. However, the
potential for reversion to virulence or vaccine-induced disease
exists. Vaccine-induced disease is most likely to occur in highly
immunosuppressed animals. Attenuated live vaccines also
have the potential to cause some immunosuppression in their
119
120 SECTION 3  Basic Principles for Infection Control
TABLE 12-1
Advantages and Disadvantages of Attenuated Live and Inactivated Vaccines
Attenuated Live
Advantages Rapid onset of immunity
Sustained immunity after single dose
May immunize others in populations
Improved breakthrough of maternal antibody
interference
Disadvantages Potential for reversion to virulence
Virulence in the immunocompromised
Contraindicated in pregnancy
May cause immune suppression
Can interfere with development of immunity if administered
within days to 2 weeks of another vaccine
Less stable in storage
Potential for vaccine contamination
own right,11,12 or they may shift the balance from Th1 to Th2
immune responses.13 Rarely, this can lead to clinical disease.
For example, an outbreak of salmonellosis was reported in cats
after use of a high-titered attenuated live FPV vaccine.14 Very
rarely, contamination of attenuated live vaccines has occurred
with other pathogenic microorganisms present within cell cul-
tures used to propagate the vaccine.
Generally speaking, inactivated vaccines are less effective
than attenuated vaccines, because replication in the host does
not occur. They produce weaker immune responses of shorter
duration, and more frequent booster immunizations may be
required. Two initial doses of vaccine 3 to 4 weeks apart are
essential to produce an effective immune response, and if more
than 6 weeks elapses between these doses, it has been recom-
mended that the series should be repeated.15 Beyond the initial
vaccination series, it is not clear whether lapsed annual boosters
require the series to be restarted. This is not considered neces-
sary for human immunization16 but has been suggested for dogs
when more than 2 or 3 years elapses between boosters.15 Inacti-
vated vaccines usually contain adjuvant as well as a large infec-
tious dose to improve immunogenicity. They are safer than live
attenuated vaccines for use during pregnancy and in very young
or debilitated animals. Although bacterins have traditionally
been associated with a greater likelihood of allergic reactions
than live attenuated vaccines, newer inactivated vaccines are
safer and have reaction rates that more closely approach those
of live attenuated vaccines. The maximum duration of immunity
that is induced by commercially available bacterins for dogs and
cats remains largely unknown, partly because challenge studies
that evaluate long-term duration of immunity are prohibitively
expensive. However, some inactivated viral vaccines have been
shown to have durations of immunity in excess of 7 years in
cats.17 Caution is required when extrapolation is made from
the duration of immunity for one product to that for a similar
product from a different manufacturer, because it may not be
equivalent. Although bacterins usually do not protect all animals
from infection, they may prevent clinical illness. In some cases,
natural infection of vaccinated animals serves to further boost
the immune response, and this can influence duration of immu-
nity in the field. The advantages and disadvantages of attenuated
live and inactivated vaccines are shown in Table 12-1.
Inactivated
Safe, even in immunocompromised and pregnant
animals
Do not interfere with development of ­immunity
from other vaccines
Stable in storage
Slow onset of immunity
Multiple boosters required
Often highly adjuvanted, with greater potential
for adverse effects
Reduced degree of protection compared with at-
tenuated live vaccines
Poor breakthrough of maternal antibody
­interference
Subunit vaccines contain specific structural components of a
microbe that stimulate a protective immune response, together
with adjuvant. They contain reduced amounts of foreign protein,
which minimizes the potential for hypersensitivity reactions.
Recombinant DNA vaccines are created through manipu-
lation of the DNA of a pathogen in the laboratory, with the
negation of pathogen virulence. Sometimes this also can allow
diagnostic tests to differentiate naturally infected from vacci-
nated animals (DIVA), because of differences in the antibody
response evoked by the vaccine. There are several different types
of recombinant DNA vaccines:
1. Recombinant subunit vaccines. These are produced by
cloning one or more genes for a protective antigen into an
expression vector, such as in Escherichia coli. The protein
expressed by the bacteria is then purified and used in the vac-
cine ­(Figure 12-1, A). An example of a recombinant subunit
vaccine is the Lyme recombinant OspA vaccine.
2. Deletion mutant vaccines. These are produced by deleting
virulence genes from a pathogen while protective antigens
are left in place. There are currently no such vaccines for
dogs and cats.
3. Vectored vaccines. These are produced by inserting genes for
one or more protective antigens into the genome of a virus.
The virus replicates in the host and expresses the antigens
but is nonpathogenic (see Figure 12-1, B). Currently avail-
able vectored vaccines for dogs and cats use canarypox virus
as a vector.
4. DNA vaccines. These consist of naked DNA that encodes
the antigens required for protective immunity. The DNA is
injected directly to the animal using an inoculation system.
The DNA is then taken up by host cells and translated into
antigen. Both humoral and cell-mediated immune responses
are produced. DNA vaccines are not currently available
commercially for use in dogs and cats.
Vaccine Storage, Handling,
and Administration
Vaccines should be stored and administered according to label
recommendations. Inactivation of vaccines can occur if they
 CHAPTER 12  Immunization 121

A B

FIGURE 12-1  Examples of recombinant DNA vaccines. A, Recombinant subunit vaccine. The gene of interest is inserted into an expression vector such as a plasmid taken up by
Escherichia coli, which subsequently produces large amounts of an immunogenic protein. This is purified and used in the vaccine. B, Vectored vaccine. The gene or genes of interest are
inserted into a canarypox or vaccinia vector, which is then inoculated into an animal. Replication of the vector within the host is followed by expression of the immunogenic protein.

are inadvertently frozen or heated to excessive temperatures,
exposed to excessive amounts of light, or used beyond their
expiration date. Hands should be washed before preparation
and administration of the vaccine. Lyophilized products should
be reconstituted with the proper diluent, and different vaccines
should not be mixed in the same syringe or vial. Reconstituted
products should be used immediately. It has been recommended
that attenuated live vaccines be discarded if more than 1 hour
has lapsed since reconstitution,15 although no published reports
exist of the viability of vaccine organisms over time after recon-
stitution or of the ability of stored, reconstituted vaccine to elicit
an immune response. Vaccines should only be used in the ani-
mal species for which they are labeled, or serious adverse effects
or failure of immunization can occur.
If vaccines for multiple different pathogens are to be admin-
istered simultaneously, they should be injected at distant sites
or, if possible, a combination vaccine should be used. Simul-
taneous vaccination for more than one pathogen does not
appear to interfere with immune responses to each component
of the vaccine,18-20 and vaccine manufacturers must demon-
strate that the protection that occurs for a specific pathogen
after vaccination with a combination product equals the pro-
tection that occurs when a vaccine for only that pathogen is
given. In contrast, successive parenteral administration of dif-
ferent attenuated live vaccines at 3 to 14 day intervals has the
potential to interfere with immune responses. An interval of
4 weeks is preferred for human patients.16,21 Inactivated vac-
cines do not produce interference in this way.16 If possible,
administration of vaccines to animals that are under anesthesia
should be avoided because adverse reactions may be difficult
or impossible to recognize in this situation. It is not necessary
to re-administer an intranasal vaccine if the animal coughs or
sneezes after administration.
The site and route of administration, product, serial number,
expiry date, and individual who administered the vaccine should
be recorded for each vaccine administered.2 Vaccine vials often
possess adhesive labels that can be easily removed and applied
to a paper medical record.
Components of the Immune Response
The immune response is divided into innate and adaptive
immune responses. The innate immune response is nonspecific
and acts as an immediate line of defense against an infection.
Components of the innate immune response consist of natu-
ral killer cells, which recognize host cells that are infected by
viruses; complement, which is activated by bacterial cell wall
components; and phagocytes, such as macrophages and den-
dritic cells. The adaptive immune response develops over sev-
eral days and involves presentation of antigen by dendritic
cells in association with the major histocompatibility complex
and stimulation of B and T cell responses, together with the
122 SECTION 3  Basic Principles for Infection Control
BOX 12-1
Factors That Can Affect Immune Responses to Vaccines
Target pathogen (e.g., respiratory versus systemic pathogen)
Vaccine composition (e.g., inadequate adjuvant)
Route of administration
Young age
Breed/genetic factors
Nutrition
Pregnancy status
Concurrent moderate to severe illness
Fever
Immunosuppressive drugs
Presence of maternal antibody
Improper vaccine storage and administration
Vaccination with an attenuated live viral vaccine within
last 3 days to 2 weeks
Inadequate time allowed for immunization before expo-
sure to field organisms
formation of memory B cells. The nature of the innate response
influences the subsequent adaptive response. Cells of the innate
immune system possess pattern recognition receptors that can
recognize patterns that are characteristic for various pathogens
(pathogen-associated molecular patterns, or PAMPs), including
Toll-like receptors and NOD-like receptors. PAMPs are under
investigation for use as adjuvants in human and animal vaccines
in order to create improved T cell immune responses.4,22
Determinants of Immunogenicity
All vaccines that are available for dogs and cats induce cell-
mediated immunity (CMI) with induction of immunologic mem-
ory and a booster effect on repeat administration. Although the
presence of antibody correlates with protection for some patho-
gens, such as CDV and CPV, a lack of antibody does not infer
a lack of protection, because of the presence of CMI, which is
more difficult to measure.
Vaccines rarely protect all vaccinated individuals from infec-
tion and disease. In particular, vaccines for canine and feline
respiratory pathogens do not prevent disease but can reduce the
prevalence and severity of disease as well as reduce the number
of organisms shed. Limited immunity following vaccination is
especially likely for infections for which immunity after natural
infection is partial or short-lived.
The ability of a vaccine to induce an immune response
depends not only on the target pathogen, vaccine composition,
and route of administration, but also on host factors such as
age, nutrition, pregnancy status, stress, concurrent infections,
and immune status, including the presence or absence of pas-
sively acquired antibody (Box 12-1). Some of these factors may
also influence vaccine safety. Some animals, particularly dogs of
the Rottweiler breed, may have an impaired ability to respond
to vaccination. These dogs have been termed nonresponders.2,23
This situation is probably rare if efficacious vaccines are used
and booster vaccines are administered. Young dogs, less than
1 year of age, have a significantly reduced response to vacci-
nation with rabies virus vaccines when compared with adult
dogs.24 Small-breed dogs have a greater serologic response to
rabies vaccines than large-breed dogs.25 Administration of vac-
cines to febrile animals or animals with moderate to severe ill-
ness should be avoided if possible until recovery has occurred,
because the immune response to the vaccine may be suboptimal.
Failure of immunization can result from an inadequate dose
of antigen. Thus, division of a single vaccine dose for adminis-
tration to a larger number of dogs and cats, or small-breed dogs
as opposed to large-breed dogs, may lead to failure of immuni-
zation. Veterinarians should not split vaccine doses because this
shifts the liability from the vaccine manufacturer to the veteri-
narian if vaccine failure occurs. Immunization can also fail in
the face of an overwhelming challenge dose.
The route of administration can influence the type of immune
response generated. Subcutaneous administration is associated
primarily with an IgG response, and rarely induces high levels of
secretory IgA antibodies. In contrast, intranasal administration
results in an IgA and, to a lesser extent, an IgG response. Immu-
nogenicity and safety may be compromised when a vaccine is
administered using the incorrect route.
In young animals, MDA can neutralize vaccine antigens and
interfere with effective immunization. This is one of the most
common reasons for vaccine failure in dogs and cats. Any MDA
titer against CPV has the potential to interfere with immuniza-
tion. The amount of MDA in a puppy or kitten at any one point
in time cannot be predicted because it varies depending on the
titer of the dam and the amount of colostrum ingested after
birth. As a result, a series of vaccinations are administered in
order to increase the chance that successful immunization will
occur soon after the decline of MDA titers to sufficiently low
concentrations (Figure 12-2). Nevertheless, a window always
exists when MDA concentrations are high enough to interfere
with immunization, but not sufficient to prevent natural infec-
tion. This window is known as the window of susceptibility or
the window of vulnerability. The use of recombinant vectored
vaccines can overcome the interference by MDA, although the
extent to which this applies in animals that have passive immu-
nity to the vector virus (i.e., immunity transferred from a dam
that was immunized with a recombinant vector vaccine) requires
clarification. Because replication of the vector is aborted, the
immune response to the vector itself may be reduced. As a result,
passive transfer of neutralizing antibody titers to the vector may
not occur. Mucosal vaccines can also provide greater protec-
tion in the face of MDA; the mucosal immune system matures
shortly after birth.26,27
Whenever possible, animals should be isolated until sufficient
time has elapsed for proper immunization. For most parenteral
and mucosal vaccines, this is 1 week (and at the absolute mini-
mum, 3 days) after inoculation. Vaccine failure can also occur
in animals that are incubating the disease for which vaccination
is performed at the time of vaccination.
Measurement of the Immune Response
For some vaccines, such as rabies, CDV, CPV, and FPV, the
presence of circulating antibodies correlates with protection
(Table 12-2). Thus, serologic assays have been used in dogs and
cats to decide whether vaccination is necessary or likely to be
effective. These serologic assays have also been used to clear
pets for travel.
Although tests that measure antibody responses in dogs
and cats have improved in recent years, different laboratories
can report significantly different values for the same serum
 CHAPTER 12  Immunization 123

FIGURE 12-2  Influence of maternal antibody (MDA) on immunization. Puppies and kittens acquire variable amounts of MDA transplacentally and through colostrum after birth. This
binds to vaccine antigens and inhibits the immune response. A series of vaccines are administered to maximize the chance of inducing an immune response as MDA concentrations decline.
The window of susceptibility is the period of time when MDA concentrations are high enough to interfere with immunization, but not sufficient to prevent natural infection. High antigen
mass vaccines provide protection earlier than low mass vaccines. (From Greene CE, Schultz RD. Immunoprophylaxis. In: Greene CE, ed. Infectious Diseases of the Dog and Cat, 3 ed. St Louis,
MO: Saunders; 2006.)

to develop disease after challenge, possibly because of over-

TABLE 12-2 whelming exposure or immune suppression. Measurement of
Antibody Titers That Correlate with Protection against antibody titers may be considered for animals that have had
Distemper, Parvovirus, and Rabies previous adverse responses to vaccination, particularly suscep-
tible breeds (e.g., Rottweilers and CPV infection). The World
Minimum Small Animal Veterinary Association (WSAVA) has suggested
Pathogen Protective Titer Methodology Used that puppies could be tested at least 2 weeks after the final
puppy vaccine to decide whether further vaccination for CDV
Canine distemper ≥1:16 to 1:20 Serum neutralization or CPV is necessary.2 Negative titers should prompt additional
virus (SN) vaccination for these puppies.
Canine parvovirus ≥1:80 to 1:100 Hemagglutination Rapid in-house serologic assays have also been used to make
inhibition (HI) decisions regarding isolation or euthanasia in shelter situations,
Rabies ≥0.5 IU/mL Fluorescent antibody through identification of immune animals.28 Unfortunately, it is
virus neutralization not always be possible to know if positive titers represent recent
(FAN) infection, and animals that test positive may still shed virus
and pose a risk to other animals. In young puppies and kittens,
positive results may represent persistent MDA or the presence
specimen, and there is a lack of validated sensitivity and speci- of active immunity, and MDA does not have the same ability
ficity for these assays. Sometimes use of these assays increases to protect against infection. In-house serologic assays can also
costs significantly and delays immunization. In-practice assays be used to decide whether pregnant animals are susceptible in
also are available for detection of antibody responses, and these a shelter environment and thus minimize adverse reactions to
have the potential to overcome problems associated with labo- attenuated live vaccines in this group of animals (see Appendix
ratory quality control and delays in immunization. In-practice I). A study that evaluated the performance of one ELISA assay
assays are generally not quantitative. Although high antibody (Synbiotics TiterCHEK CPV/CPV) found that for CPV antibod-
titers are generally associated with greater protection, an ani- ies, the sensitivity and specificity of the assay was 92% and 94%,
mal with no titer may still be resistant to challenge because respectively, when compared with hemagglutination inhibition;
of CMI, which is not measured. Conversely, an animal with a and for CDV was 76% and 92%, respectively, when compared
titer that is generally regarded as protective has the potential with serum neutralization.29
124 SECTION 3  Basic Principles for Infection Control
FIGURE 12-3  Facial edema and hyperemia in a 4-month old intact male Chihuahua
mix after vaccination with a bacterin vaccine. (Courtesy Dr. Stephen D. White, University of
California, Davis Veterinary Dermatology Service.)
Adverse Reactions to Vaccines
A vaccine is needed if an infectious disease causes significant
morbidity and mortality. Vaccines should prevent more disease
than they cause. In order to produce protective immunity, a vac-
cine must stimulate a reaction in an animal, both at the site of
injection and systemically. This may cause clinical signs. Ide-
ally, the signs are mild and either unnoticeable or acceptable to
the pet owner. In rare situations, adverse reactions are severe
enough to threaten a pet’s life. Sometimes, enhanced efficacy
leads to a reduction in vaccine safety. Veterinarians are encour-
aged to report adverse reactions to vaccines to a technical service
veterinarian employed for this purpose by the vaccine manufac-
turer. In some countries, the drug company then reports details
of the adverse reaction to drug regulatory authorities. An under-
standing of the true nature and incidence of adverse effects
associated with vaccination has been hampered by underreport-
ing and variable delays between vaccination and the inconsis-
tent appearance of potential, more chronic systemic adverse
effects.30 In addition, correlation of adverse reactions to vaccine
administration in young animals may be difficult because of the
uniform and frequent administration of vaccines to this group.
For example, it has been difficult to prove a connection between
vaccination for distemper and development of hypertrophic
osteopathy in Weimaraner dogs.
Immune-Mediated Vaccine Reactions
Type I Hypersensitivity Reactions
Type I hypersensitivity reactions occur when allergens cross-
link IgE molecules that are bound to receptors on mast cells
and basophils and trigger degranulation. Clinical signs of type
I hypersensitivity responses that occur after vaccine administra-
tion include facial or periorbital edema, urticaria, cutaneous
hyperemia, generalized pruritus, salivation, hypotensive shock,
tachypnea, vomiting, diarrhea, collapse, and even death (Figure
12-3). Vomiting and respiratory distress are common in cats.
These signs generally occur within 24 hours of vaccine admin-
istration; anaphylaxis usually begins within minutes. The esti-
mated incidence of anaphylaxis after vaccination of dogs and
cats is 1 in 5000 to 1 in 50,000 and depends on the vaccines
used. One retrospective study evaluated 1.23 million dogs and
nearly 0.5 million cats from more than 300 Banfield hospi-
tals in the United States in 2002 through 2005. In this study,
vaccine-associated adverse effects that were listed as vaccine
reactions, allergic reactions, anaphylaxis, urticaria, and/or car-
diac arrest were documented within 3 days of vaccine adminis-
tration in 38.2 per 10,000 dogs and 47.4 per 10,000 cats.20,31
Reactions coded as “allergic” or “anaphylaxis” were reported
in approximately 1 in 785 dogs and 1 in 1200 cats. Reactions
coded as anaphylaxis constituted only 5% of these reactions.
Death occurred in 1 in 400,000 dogs and 1 in 125,000 cats that
received vaccines, and all 3 dogs and 1 of the 4 cats that died
received four or more doses of a vaccine (i.e., more than one
vaccine product administrated simultaneously). Most reactions
in dogs (73%) occurred on the day the vaccine was administered
(day 0), 19% occurred on day 1, 6% on day 2, and 3% on
day 3. Data from the UK Veterinary Products Committee report
indicated anaphylaxis in 1 in 385,000 vaccinated dogs and 1 in
555,000 cats.23
Vaccines that contain large amounts of adjuvant, certain pre-
servatives, or inactivated bacteria with proinflammatory outer
surface components are more likely to cause reactions. Proteins
present in fetal calf serum and stabilizers such as gelatin within
the vaccine may also be responsible for allergic reactions.32
In the Banfield study, the risk of reactions increased with the
number of vaccine doses (i.e., volume of vaccine in milliliters)
administered per office visit.20 Small-breed dogs, such as minia-
ture dachshunds, pugs, Boston terriers, miniature pinschers, and
Chihuahuas, were more susceptible to development of acute vac-
cine reactions, and the risk of a vaccine-related adverse increased
as body weight decreased. The risk of vaccine-related adverse
events was 4 times greater in dogs that weighed 5 kg or less than
in those that weighed more than 45 kg (Figure 12-4). Adverse
events increased in frequency with age up until 2 years of age in
dogs and 1 year of age in cats, after which the frequency progres-
sively declined to rates lower than that observed in animals less
than 1 year of age (see Figure 12-4). The decrease in frequency
with older age may have occurred because of owners’ unwilling-
ness to have their pets vaccinated if a previous reaction occurred.
Sexually intact dogs were less likely to develop adverse reac-
tions than neutered dogs, but the opposite was true for cats.20,31
Female cats were more likely to exhibit reactions than male cats.
The treatment of choice for anaphylaxis is epinephrine,
together with other supportive treatments such as intravenous
fluids and supplemental oxygen if necessary. Antihistamines
and corticosteroids can be administered to dogs with less severe
reactions. Vaccination should be avoided in animals with a his-
tory of severe reactions. Pretreatment with an antihistamine
could be considered in animals with a history of mild reactions.
These animals should also be monitored closely in the hospital
for several hours after vaccine administration. It has been sug-
gested that in the future, commercial production of low-dose
vaccines for small-breed dogs might be more appropriate, given
their increased risk of reactions and more marked serologic
responses to vaccination.33
Other Hypersensitivity Reactions, including Autoimmune Disease
Type II hypersensitivity reactions occur when IgG and IgM bind
to cell surface antigens and fix complement, with target cell lysis
or removal of target cells by macrophages within reticuloen-
dothelial tissues. Concerns have been raised that vaccination
may predispose certain genetically susceptible individuals to
immune-mediated cytopenias, although as in human medicine,
studies of dogs and cats to date have failed to conclusively docu-
ment vaccines as causes of these and other chronic diseases.21,34
 CHAPTER 12  Immunization 125

Immune-mediated hemolytic anemia was suspected to occur

A uveitis and subclinical nephritis developed in 0.4% of dogs
B immune-mediated polyarthritis in some dogs,43 but this associa-
FIGURE 12-4  Correlation between (A) body weight and (B) age and the occurrence
of vaccine-associated adverse effects within 3 days of vaccination in dogs. (From Moore GE,
Guptill LF, Ward MP, et al. Adverse events diagnosed within three days of vaccine adminis-
tration in dogs. J Am Vet Med Assoc 2005;227(7):1102-1108.)
following parvovirus vaccination, possibly due to the hemag-
glutinating properties of the virus and the high antigen mass
in some of these vaccines,35 but a later retrospective case-­
control study found no association.36 Transient thrombocyto-
penia can occur after vaccination in some dogs.37 In one study,
dogs developed antithyroglobulin antibodies after vaccination,
although this was not associated with development of hypothy-
roidism.38 Cats that are vaccinated with Crandell-Rees feline
kidney (CRFK) cell-derived vaccines develop antibodies against
the CRFK proteins alpha-enolase and annexin A2.39 Whether
production of these antibodies has clinical significance remains
to be determined.
Type III hypersensitivity reactions are characterized by
immune-complex deposition in tissues and may be a consequence
of immunization with certain vaccines. For example, anterior
receiving the canine adenovirus-1 (CAV-1) vaccine. This vaccine
has now been replaced by CAV-2 vaccines, which rarely produce
these lesions. A cutaneous vasculitis has been described after
vaccination of dogs and cats with rabies virus vaccines (“rabies
vaccine-induced vasculitis”)40-42 (Figure 12-5). This can occur at
the site of vaccine administration, and in some animals, a mul-
tifocal ischemic dermatopathy and myopathy that affects sites
such as the pinnal margins, periocular areas, tail tip, and paw
pads has been reported to occur 1 to 5 months after the appear-
ance of the initial skin lesion.40 The multifocal dermatopathy
and myopathy has been reported to resolve after treatment with
pentoxifylline and vitamin E. Additional evidence is required
to strengthen the association between the multifocal condition
and rabies vaccination. Similarly, concerns have been raised
about a possible temporal association between vaccination and
tion remains unproven. Immune-mediated polyradiculoneuritis,
a type IV hypersensitivity reaction (delayed-type hypersensitiv-
ity), occurred after vaccination of dogs with suckling-mouse
brain-derived inactivated rabies vaccines, which are no longer

A B
FIGURE 12-5  Ischemic dermatopathy suspected to be associated with rabies virus vaccination that involved the pinnae (A) and footpads (B) of a 1-year-old male dachshund.
The rabies vaccine was administered several months before the onset of signs. (Courtesy Dr. Stephen D. White, University of California, Davis Veterinary Dermatology Service.)
126 SECTION 3  Basic Principles for Infection Control
available.44 Subsequent reexposure resulted in more severe and
prolonged paralysis. Granuloma formation at the site of vaccine
administration also represents a type IV hypersensitivity reaction.
Despite the lack of conclusive evidence for an association
between vaccination with currently available vaccines and auto-
immune disease, it is possible that vaccination may be associated
with dysregulation of the immune response in predisposed indi-
viduals. Therefore, vaccination is often withheld if not absolutely
necessary in dogs and cats with a history of autoimmune disease.
Serum titers could also be assessed to gauge the need for specific
immunization in these animals.
Vaccine Organism-Induced Disease
Disease occasionally results from replication of microorganisms
present in a vaccine, although severe disease is uncommon with
currently available vaccines. Fever and lethargy are the most
common adverse effects of vaccination and result from cytokine
production in response to the vaccine. These are transient and
usually resolve within 1 to 2 days.
In the past, the use of attenuated rabies virus vaccines resulted
in ascending paralytic disease in a proportion of cats and dogs.
This led to a change to inactivated, adjuvanted rabies vaccines.
Administration of attenuated parvovirus vaccines to pregnant
cats and dogs can lead to cerebellar disease in the fetus, and
these vaccines have the potential to cause severe disease if shed
vaccine virus infects colostrum-deprived neonates that are less
than 2 weeks of age.2 Some CDV vaccines have been associated
with postvaccinal distemper in young puppies.45 As a result,
vaccination with live attenuated CDV and CPV (dogs) and
feline panleukopenia virus (cats) vaccines should be avoided in
pregnant animals, puppies and kittens less than 6 weeks of age,
and animals receiving potent anticancer chemotherapy drugs.
Some CDV vaccine strains, such as Rockborn-like strains, are
more virulent than others, and these may continue to circulate
and contribute to distemper in the dog population.46 The safest
attenuated live CDV vaccines contain the Onderstepoort strain.
Vaccination of certain exotic pet, zoo, and wild animal species,
such as ferrets, with any attenuated live CDV vaccine for dogs
can also lead to postvaccinal distemper.47,48 For animals with
chronic immunocompromise, the use of inactivated vaccines has
been recommended if immunization is deemed necessary. How-
ever, inactivated vaccines may have reduced efficacy in immuno-
compromised animals compared with healthy animals.
The use of mucosal (e.g., intranasal) vaccines for respiratory
pathogens in dogs and cats can be followed by development of
mild to moderate, transient upper respiratory tract signs. There
have been concerns that mucosal Bordetella bronchiseptica
vaccines may cause respiratory disease in immunosuppressed
humans who inhale the vaccine directly during administration
or who contact vaccine organisms that are subsequently shed
by the vaccinated dog,49,50 but definitive proof of this is still
required. Inadvertent parenteral administration of the aviru-
lent live intranasal B. bronchiseptica vaccine to dogs can lead
to local injection-site reactions and, occasionally, fatal hepatic
necrosis.51 Inadvertent parenteral administration of mucosal
B. bronchiseptica vaccines should be treated with subcutane-
ous fluids at the site of administration and treatment with an
oral antibiotic likely to be effective against B. bronchiseptica,
such as doxycycline. The ASPCA Poison Control Center also
recommends injection of a gentamicin sulfate solution into
the affected area (2 to 4 mg/kg gentamicin sulfate in 10 to
30 mL of saline).4 Doxycycline treatment could be continued
FIGURE 12-6  Two-year-old female toy poodle/maltese terrier mix that developed
focal alopecia at the site of vaccination followed by regrowth of hair with an altered color
and texture. (Courtesy Nicole Pierce, University of ­California, Davis, Class of 2013.)
for 5 to 7 days. Dogs that develop hepatic necrosis may need
more aggressive supportive care.
Local Cutaneous Reactions and Injection-Site
Sarcomas
Local cutaneous reactions are common adverse effects of vac-
cination, especially in cats, and include pain, swelling, irritation,
and abscess formation. In dogs, focal alopecia or discoloration
of the haircoat at the vaccination site can also occur (Figure
12-6). Inactivated, adjuvanted vaccines have been most com-
monly incriminated. Focal cutaneous granulomas and some-
times permanent focal alopecia have been most commonly
reported after inactivated rabies vaccine administration to
breeds such as Maltese terriers and bichon frisé.
In the late 1980s, an increase in inflammatory injection-site
reactions at the site of rabies vaccine administration were noted
in canine and feline biopsy specimens sent to the University
of Pennsylvania. Shortly thereafter, sarcomas were observed
at these sites in cats in the United States, with a 25% increase
each year from 1987 to 1991 (Figure 12-7).52 This followed
(1) the change from attenuated live to inactivated rabies vac-
cines, (2) increased use of rabies vaccines in cats, and (3) the
introduction of FeLV vaccines. The national incidence of sar-
coma formation is estimated to be 0.6 to 2 sarcomas per 10,000
cats that are vaccinated.53,54 In contrast, in the United King-
dom, it was 0.21 per 10,000 vaccine doses sold between 1995
and 1999.22 The interval between tumor development and the
last rabies vaccine typically ranges from 3 months to 3.5 years.
Most tumors are fibrosarcomas, but other types of sarcomas can
also occur. Although development of injection-site sarcomas is
clearly linked to administration of FeLV, rabies, and other vac-
cines, development of sarcomas is not related to the use of spe-
cific brands or types of vaccine within an antigen class (with the
possible exception of recombinant vaccines56), reuse of syringes,
needle gauge, use and shaking of multidose vials, or concomi-
tant viral infection.55 There is also no evidence that aluminum-
containing vaccines are associated with a higher risk of sarcoma
development than aluminum-free vaccines, but there has been
concern that adjuvant stimulates an inflammatory response that
predisposes to sarcoma formation. A recent study showed that

A B
FIGURE 12-7  A, Computed tomographic image of an injection-site sarcoma over the scapula of a 12-year old male neutered domestic shorthair cat. B, Discoloration of the haircoat
after radiation therapy.
inactivated vaccines were approximately 10 times more likely to
be associated with injection-site sarcomas when administered at
the pelvic limb site than nonadjuvanted recombinant vaccines.56
However, neither attenuated live, recombinant, nor inactivated
vaccines are risk-free, and injection of certain long-acting inject-
able medications (especially glucocorticoids) can also be associ-
ated with sarcoma formation in cats years later.56,57 There is
some evidence that administration of cold vaccine may also be
more likely to be associated with sarcoma formation, but this
requires verification.55 It is currently hypothesized that an indi-
vidual cat’s genetically programmed wound healing response is
responsible for the development of injection-site sarcomas.
Treatment of injection-site sarcomas involves aggressive sur-
gical resection followed by full-course post-operative radiation
therapy, because of the high incidence of recurrence. Possible
adverse effects of radiation include mild to moderate cutaneous
burns, hypopigmentation of the hair in the field (see Figure 12-7,
B), and damage to the spinal cord, lungs, and kidneys within the
field, although the last is rare. In one study, the median sur-
vival time for cats treated with postsurgical curative radiation
therapy was 43 months.57 Most of these cats had clean margins
after surgical resection of the tumor. In contrast, the median
survival times for cats treated with coarse fractionated radiation
therapy was 24 months. These cats generally had macroscopic
disease or dirty margins. Adjuvant chemotherapy with carbo-
platin or single-agent doxorubicin also has been associated with
improved outcome.57
In order to prevent death from sarcoma formation in cats,
the Vaccine-Associated Feline Sarcoma Task Force (VAFSTF) in
North America recommended that rabies vaccines be adminis-
tered as distally as possible on the right pelvic limb, and leuke-
mia vaccines be administered as distally as possible on the left
pelvic limb (rabies right, leukemia left). Care should be taken
not to administer the vaccine too proximally or in the flank
region, because tumors in these locations cannot be resected as
effectively.58 Other core vaccines should be administered over
the right shoulder. These recommendations were not adopted by
the WSAVA, which suggested that the skin of the lateral thorax
and abdomen be used for vaccination, and vaccination sites be
rotated from year to year.2 Both groups recommended that the
interscapular region be avoided, because vaccine constituents
CHAPTER 12  Immunization 127
can pool in this region and contribute to a chronic inflamma-
tory response. In addition, both groups recommended that the
sites of administration and the product and batch number be
documented to facilitate the reporting of adverse events. Exces-
sive administration of vaccines to cats should be minimized,
and alternative routes, such as intranasal immunization, should
be considered. Owners should be advised to monitor injection
sites for 3 months after vaccines are administered. If a lump
forms and increases in size 1 month after vaccination, or persists
beyond 3 months, the owner should have the mass evaluated by
the veterinarian.
In accordance with the VAFSTF recommendations, the ana-
tomic location, shape, and size of masses that develop at injec-
tion sites should be documented, and an incisional or tru-cut
biopsy performed. Fine-needle aspiration is not recommended.
If the mass is malignant, routine laboratory tests and thoracic
radiography should be performed, together with computed
tomography or magnetic resonance imaging of the mass if client
finances allow. A veterinary oncologist should be consulted, and
if possible, referral to a specialist surgeon or oncologist is rec-
ommended. Wide excision that includes at least a 2-cm margin
is necessary, and the entire piece of tissue excised should be sub-
mitted for histopathology and evaluation of surgical margins.
Additional treatment, such as chemotherapy and radiation ther-
apy, is often necessary. Cats should be reevaluated at 3-month
intervals for a year after surgery.
Interference with Diagnostic Test Results
Vaccination has the potential to interfere with the results of
assays that detect the antibody response to infection or assays
that detect components of a pathogen itself.
Interference with antibody test results can be specific or non-
specific. Nonspecific interference is rarely identified, but results
from cross-reactivity between antibodies to vaccine components
(such as albumin) and the reagents used in serodiagnostic tests.
More commonly, specific interference with serodiagnostic tests
for the infection that is targeted by the vaccine occurs. This espe-
cially problematic if (1) vaccination does not completely protect
against infection, (2) the results of serologic tests are required
for diagnosis, and (3) infection is chronic and persistent, and so
identification of recent natural infection through seroconversion
128 SECTION 3  Basic Principles for Infection Control
is usually not possible. For example, the inactivated FIV vaccine
does not provide 100% protection, but vaccinated cats develop
antibodies to the vaccine virus. These antibodies are detected by
ELISA and Western blot immunoassays used for diagnosis of
FIV infection. In the absence of a history of vaccination, posi-
tive antibody test results indicate active infection.59 PCR can be
used to detect FIV infection in infected cats that have a history
of vaccination with FIV vaccines, but PCR can occasionally be
negative in cats with active infection, so a negative PCR result
does not rule out natural infection with FIV. Vaccine interfer-
ence with serodiagnosis can occur after vaccination of dogs for
influenza or leptospirosis, but because both of these diseases
are acute, seroconversion can be used for diagnosis of recent
infection in vaccinated dogs. Some serologic assays differentiate
between vaccinated and naturally infected animals (DIVA). For
example, serologic assays that detect the C6 antigen of Borrelia
burgdorferi do not detect antibodies that result from immuni-
zation with Lyme vaccines. The development of recombinant
vaccines that stimulate a pattern of antibody responses that dif-
fer from those that result from natural infection can help to
overcome issues related to differentiation of naturally infected
and vaccinated animals.
Interference with the results of assays that detect the patho-
gen itself (as opposed to the antibody response) occurs after
vaccination with attenuated live vaccines that are shed by ani-
mals after vaccination. For example, cats may test positive using
ELISA assays for FPV antigen after vaccination with attenuated
live FPV vaccines, although for some assays, the rate at which
this occurs is very low.60 PCR tests can be positive for extended
periods after vaccination with attenuated live vaccines. In some
cases, sequence analysis of the PCR product can sometimes
allow differentiation between vaccine and field strains, but
this is currently performed only on a research basis.61 Rapid
PCR assays have also been designed that differentiate between
vaccine and field strains of some pathogens, such as CDV or
B. bronchiseptica.62,63 Quantification of organism numbers
present in a specimen through the use of real-time PCR assays
(e.g., for CDV) may shed light on whether natural infection
(high organism load) or vaccination (low organism load) has
occurred.
Vaccine Selection
The advantages and disadvantages of vaccines and vaccine
combinations that are currently available on the market are
provided in the relevant sections of this book for each infec-
tion entitled “Immunity and Vaccination.” Suggested vaccina-
tion schedules for individual pets and shelter animals that are
based on recommendations provided by the American Animal
Hospital Association (AAHA), the American Association for
Feline Practitioners (AAFP), the European Society for Feline
Medicine (ESFM), and WSAVA are summarized in tables in
Appendix.2,15,64-73
To facilitate vaccine selection, vaccines for dogs and cats have
been divided by various task forces into core vaccines, noncore
vaccines, and those that are generally not recommended.
Core vaccines are recommended for all animals with an
unknown vaccination history. The diseases involved have sig-
nificant morbidity and mortality and are widely distributed, and
in general, vaccination results in good protection from disease.
All shelter animals should be vaccinated with core vaccines
before entry to a shelter or at the time of entry if immunization
ahead of time is not possible. Canine core vaccines include
vaccines for CPV, CDV, CAV, and rabies for countries where
rabies is endemic. The core feline vaccines are those for feline
­herpesvirus-1 (FHV-1), feline calicivirus (FCV), FPV, and rabies.
Noncore vaccines are optional vaccines that should be con-
sidered in light of exposure risk, that is, based on geographic
distribution and the lifestyle of the pet. Vaccines considered
as noncore vaccines for dogs are canine parainfluenza virus,
canine influenza virus, B. bronchiseptica, Leptospira spp.,
and Borrelia burgdorferi. Optional or noncore vaccines for
cats include FeLV, FIV, virulent FCV, Chlamydia felis, and
B. bronchiseptica vaccines.
Several other vaccines are currently available on the mar-
ket. For dogs, these are vaccines for canine coronavirus, CAV-
1, and rattlesnake envenomation. The reports of the American
Veterinary Medical Association (AVMA) and the AAHA canine
vaccine task force have listed the first two vaccines as not gen-
erally recommended, because “the diseases are either of little
clinical significance or respond readily to treatment,” evidence
for efficacy of these vaccines is minimal, and they may “produce
adverse events with limited benefit.” Currently, information
regarding the efficacy of the canine rattlesnake vaccine is insuf-
ficient. For cats, the feline infectious peritonitis (FIP) vaccine is
not generally recommended by the AAFP.
SUGGESTED READINGS
Baker C, Pickering L, Chilton L, et al. General recommendations on immu-
nization—recommendations of the Advisory Committee on Immuniza-
tion Practices (ACP). MMWR Recomm Rep. 2011;60(2):1-64.
Day MJ, Horzinek MC, Schultz RD. Guidelines for the vaccination of
dogs and cats. Compiled by the Vaccination Guidelines Group of the
World Small Animal Veterinary Association. J Small Anim Pract.
2007;48(9):528-541.
Larson LJ, Newbury S, Schultz RD. Canine and feline vaccinations and
immunology. In: Miller L, Hurley K, eds. Infectious Disease Manage-
ment in Animal Shelters. Ames, IA: Wiley-Blackwell; 2009:61-82.
Moore GE, HogenEsch H. Adverse vaccinal events in dogs and cats. Vet
Clin North Am Small Anim Pract. 2010;40:393-407.
Paul MA, Appel M, Barrett R, et  al. Report of the American Animal
Hospital Association (AAHA) Canine Vaccine Task Force: executive
summary and 2003 canine vaccine guidelines and recommendations.
J Am Anim Hosp Assoc. 2003;39(2):119-131.
The 2006 American Association of Feline Practitioners Feline Vaccine
Advisory Panel Report. J Am Vet Med Assoc 229:1405–1441 (also
http://www.aafponline.org/resources/practice_guidelines.htm).
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 SECTION 1
Viral Diseases
CHAPTER 13
Rabies
Jane E. Sykes and Bruno B. Chomel
Overview of Rabies
First Described: Circa the second millennium bc, Mesopotamia1
Cause: Rabies virus (Family Rhabdoviridae, Genus Lyssavirus)
Primary Mode of Transmission: biting, with inoculation of
saliva containing the virus
Affected Hosts: All warm-blooded animals. Highly suscepti-
ble hosts include wolves, foxes, coyotes, jackals, dogs, cat-
tle, raccoons, skunks, bats, and mongooses. Moderately
susceptible hosts include cats, ferrets, primates, sheep,
goats, and horses.
Geographic Distribution: Classic (genotype 1) rabies is found
worldwide except for Taiwan, Australia, New Zealand, Ice-
land, the United Kingdom, Japan, most of western Europe,
Fiji, Hawaii, and Guam.
Major Clinical Signs: Fever, lethargy, behavioral changes,
pupillary dilation, ataxia, lower motor neuron paresis or
paralysis (including of the muscles of mastication), ves-
tibular signs (including a head tilt), dysphagia, dysphonia,
tremors, seizures, ptyalism.
Differential Diagnoses: Distemper virus infection; West Nile
virus infection; equine encephalitis virus infection; bacte-
rial, protozoal, or fungal meningoencephalitis; toxins such
as strychnine; central nervous system trauma; spinal neo-
plasia; granulomatous meningoencephalitis.
Public Health Significance: Rabies is a deadly zoonosis and
dogs are a major natural reservoir of the virus. In the United
States, most human rabies is associated with bat contact.
Etiologic Agent and Epidemiology
Rabies is a deadly, zoonotic, neurologic disease caused by a
bullet-shaped, enveloped RNA virus that belongs to the genus
Lyssavirus (from Lyssa, the Greek goddess of madness, rage,
132
and frenzy) (Figure 13-1). The virus is fragile in the environ-
ment, and readily inactivated by a variety of disinfectants,
soaps, ultraviolet light, and heat. It can survive up to 3 to 4 days
in carcasses at 20°C, and longer with refrigeration.2,3 Freezing
tissues at temperatures less than −20°C may prolong survival of
the virus for years.
There are seven lyssavirus genotypes (Table 13-1). Classi-
cal rabies virus belongs to genotype 1. The remaining six lys-
savirus genotypes primarily infect bats and less frequently cause
fatal human encephalitis, which is clinically indistinguishable
from classical rabies.4 Thus, although Taiwan, Australia, New
Zealand, Iceland, the United Kingdom, Japan, most of west-
ern Europe, Fiji, Hawaii, and Guam are designated terrestrial
rabies-free, lyssaviruses capable of causing fatal encephalitis
in humans exist in some of these regions, including Australia,
Western Europe, and the United Kingdom (Figure 13-2). The
remaining information in this chapter pertains only to genotype
1 rabies virus infections.
Infection with rabies virus most commonly occurs as a result
of inoculation of virus-containing saliva into a bite wound, in
the large majority of the cases after a dog bite. Other routes
of transmission reported in species other than dogs and cats
Envelope (membrane)
Glycoprotein
Matrix protein
RNA genome
helical coil
FIGURE 13-1  Structure of a rabies virus virion.
 CHAPTER 13  Rabies 133

TABLE 13-1
Lyssaviruses Belonging to the Family Rhabdoviridae
Genotype Virus Source Distribution
1 Rabies virus Dog, fox, raccoon, bat, others Widespread
2 Lagos bat virus Bats, cats, not in human beings Africa (rare)
3 Mokola Shrews, cats, dog Africa
4 Duvenhage Insectivorous bats Africa (rare)
5 and 6 European bat lyssavirus Insectivorous bats Netherlands, United Kingdom, Denmark,
types 1 and 2 Germany, Spain, France, Poland, Hungary,
Russian Federation, Ukraine, Switzerland
7 Australian bat lyssavirus Flying fox (fruit bats); insectivorous bats Australia; Philippines (serologic evidence)

FIGURE 13-2  Global distribution of lyssaviruses. Distribution of rabies virus (red). Areas where rabies-related viruses only, and not classic rabies genotype 1, are documented are clas-
sified by the WHO as “rabies free” (dark blue). White areas are free of rabies and rabies-related viruses. (Redrawn from Warrell MJ, Warrell DA. Rabies and other lyssavirus diseases. Lancet
2004;363:959-969.)

are rare and include corneal or solid-organ transplantation in
human patients5; aerosol transmission, such as that occurring
within caves containing large numbers of bats6,7; and transmis-
sion after ingestion of infected tissues or milk.6,8
All warm-blooded animals are susceptible to rabies, although
dogs, wild carnivores, and bats are considered the main natural
reservoirs of rabies virus. In general, wolves, foxes, coyotes,
jackals, dogs, cattle, raccoons, skunks, bats, and mongooses
are highly susceptible to infection. Moderately susceptible hosts
include cats, ferrets, primates, sheep, goats, and horses. Marsu-
pials, including opossums, have a low degree of susceptibility.
Some bird species can be experimentally infected but do not
develop clinical signs and are unlikely hosts.9
Not every bite from a rabid animal leads to rabies virus infec-
tion, and infection may not always culminate in death, unless
clinical signs develop. Factors influencing the outcome after a
134 SECTION 1  Viral Diseases
A B
FIGURE 13-3  A, Reported cases of rabies that involve cats and dogs, by county, 2010. Histogram represents numbers of counties in each category for total number of cats and dogs
submitted for testing. Note the overlap with the distribution of wildlife reservoir hosts in B. B, Distribution of major rabies virus variants among mesocarnivore reservoirs in the United
States. (A and B modified from Blanton JD, Palmer D, Dyer J, et al. Rabies surveillance in the United States during 2010. J Am Vet Med Assoc 2011;239:773-783.)
bite from a rabid animal include the proximity of the bite site
to the central nervous system (CNS), the degree of innervation
of the bite site, the age of the host (young animals are more
susceptible), the amount of virus inoculated, and the neuroin-
vasiveness of the rabies virus variant involved. If left untreated,
up to 60% of humans bitten by a rabid dog on the head or neck
develop rabies, compared with up to 40% of those bitten on the
hand, and 10% of those bitten on the trunk or leg.10
Rabies viruses that circulate within a certain geographic
region are adapted to specific dominant reservoir hosts (e.g., bat
rabies virus variants or raccoon rabies virus variants). All rabies
virus variants can infect and cause disease in animals other than
their reservoir hosts (spillover hosts). In all host species, rabies
is usually an acute fatal illness, although some animals, such as
bats and skunks, have a more prolonged course of illness than
others, such as foxes, coyotes, or raccoons. The possibility of a
subclinical carrier state, especially in bats, has been suggested
based on the detection of rabies virus antibody and, rarely, the
virus itself in some reservoir host species that are not showing
signs of illness.4
Over the past 50 years, owing to mandatory vaccination and
control programs, domestic animal rabies in the United States has
decreased in prevalence, whereas wildlife rabies has increased.
The dog rabies virus variant was considered eradicated from the
United States at the beginning of the 21st century. Currently,
cats are more commonly reported with rabies in the United
States (about 300 cases/year) than dogs (fewer than 100 cases/
year), most likely reflecting rabies vaccination practices ­(Figure
13-3, A). No feline rabies virus variant has been described.
The geographic distribution of feline and canine rabies fol-
lows that of regions where wildlife rabies is endemic. Most
cats and dogs are infected with the rabies virus variant that is
associated with the major wildlife reservoir host in their respec-
tive geographic location (see Figure 13-3, B). Rabid cats are
most often reported from the northeastern United States, mid-
Atlantic, and south-Atlantic states, with lesser numbers from
the north and south central states. The eastern distribution
of feline rabies follows the distribution of raccoon rabies.11,12
Rarely, cats are infected from bats. Rabies in dogs and cats is
most commonly reported from the north central, south central,
and Atlantic states (see Figure 13-3, A). Dogs may be infected
with rabies virus variants from skunks or raccoons. Infected
dogs have also been imported into the United States from other
countries where canine rabies variants are endemic.13,14 In
Europe, the major wildlife reservoir species have been foxes
and raccoon dogs, and in South Africa, jackals and mongooses
predominate. In western Europe, fox rabies has largely been
eliminated as a result of mass vaccination campaigns. Spillover
occurs from wildlife reservoir hosts to domestic animal species,
which subsequently contact humans. Nevertheless, the vast
majority of human cases in North America result from contact
with infected bats, which are present in all states except Hawaii
(see Public Health Aspects). Rabies in rodents and lagomorphs
is rare, possibly because they are always killed by the bite of
a rabid animal. In the United States, most reports have been
from woodchucks in raccoon endemic areas.15 A few cases of
rabies in pet ferrets, rabbits, and a pet guinea pig have been
described.16
Although rabies occurs in cats and dogs of any age, it is most
frequently reported in young dogs and cats, with a median age
of 1 year for both cats and dogs in one study; 75% of animals
were 3 years of age or younger.17 Rabies can occur in puppies
and kittens that have not yet reached the age at which routine
rabies vaccination is approved, which is an emerging issue as a
result of illegal importation of pet dogs from Mexico through
California and Texas. Male dogs may be slightly more pre-
disposed, and most dogs and cats with rabies have not been
neutered and live in rural environments, frequently being unre-
strained at night.11,17 In the United States, some degree of sea-
sonality occurs for rabies in dogs and cats, which correlates
with seasonal increases in rabid raccoons and skunks.11,17 For
the majority of cats and dogs with rabies, a history of known
contact with wild animals or a wound is lacking.
Clinical Features
Signs and Their Pathogenesis
After inoculation into the subcutaneous tissues and muscle,
rabies virus replicates locally within muscle cells and then
attaches to peripheral nerve endings. Local replication around
the bite site can continue for months before the virus enters
peripheral sensory and motor nerve endings, with the nicotinic
acetylcholine receptor being the main receptor for the virus. The
virus then travels in a retrograde fashion up nerve axons at a
 CHAPTER 13  Rabies 135

occur. Unfortunately, rabies is infrequently suspected as the

TABLE 13-2 possible diagnosis at the time rabid animals are examined by
Major Clinical Findings in 183 Cats and 119 Dogs with Rabies veterinarians.11,17
When it occurs, the prodromal phase lasts 2 to 3 days in dogs
in the United States
and 1 to 2 days in cats, and is characterized by a variable fever,
licking or chewing at the bite site, and behavioral changes. Dogs
Sign Percent of Cats Percent of Dogs
and cats may become lethargic, anorexic, apprehensive, restless,
Aggression 55 31 or reclusive, and vomiting may occur.11 Some animals become
Ataxia 30 49 more docile or affectionate. Pupillary dilation, sometimes with
Irritability 34 23 decreased pupillary light reflexes, may occur. The furious
phase occurs in approximately two thirds of affected cats and
Anorexia 22 38 dogs22 and lasts 0 to 7 days. Clinical signs result from forebrain
Lethargy 20 37 involvement and include irritability, anxiousness or excitabil-
Hypersalivation 14 41 ity, hyperesthesia, hypersalivation, vocalization, roaming, and
aggression. Affected animals may try to eat foreign objects,
Dysphagia 10 30
which may become lodged within the gastrointestinal tract, or
Lameness 18 16 they may attack their surroundings or moving objects. Some
Limb paralysis 17 29 animals develop ataxia, vestibular signs, and grand mal sei-
Jaw paralysis 3 29 zures. Tremors; staring, or a wild, spooky, or blank look in the
eyes; increased vocalization; and compulsive running can occur
Dysphonia 16 9
cats, which also often develop aggressive behavior.11,17,23 The
Hyperesthesia 10 17 paralytic phase develops 1 to 10 days after the onset of clinical
Seizures 8 14 signs and is characterized by flaccid paralysis, which is ascend-
Fever 3 14 ing and often initially involves the bitten extremity. Neurologic
examination reveals flaccid paralysis with absent segmental
From Eng TR, Fishbein DB. Epidemiologic factors, clinical findings, reflexes. Laryngeal paralysis may lead to a change in the sound
and vaccination status of rabies in cats and dogs in the United States or pitch of a dog’s bark or cat’s meow. Hypersalivation results
in 1988. National Study Group on Rabies. J Am Vet Med Assoc from paralysis of the pharyngeal muscles, and a “dropped jaw”
1990;197:201-209. can occur as a result of masticatory muscle paralysis, especially
in dogs, which may appear to owners as if they are choking.
rate of 3 mm/hr.6 Once the virus is within the CNS, there is mas- If euthanasia is not performed, coma and death usually
sive viral replication, with cell-to-cell transmission of the virus occurs within a week of the onset of signs, with a few ani-
across synaptic junctions. The spinal cord, medulla oblongata, mals dying as long as 10 days after the onset of illness. Death
periaqueductal gray matter, limbic system, and cerebellum are is associated with multiorgan failure, especially cardiac and
particularly affected. The virus also moves outwards from the respiratory failure. Recovery from rabies is extremely rare.
CNS in somatic and autonomic nerves and is deposited in a vari- Experimentally, some dogs have recovered from clinical rabies
ety of tissues, including cardiac and skeletal muscle, the eye, the days to months after being infected with certain rabies virus
kidney, pancreas, nerves around hair follicles, and the salivary strains.24,25
glands. Production of new virions through budding from the
plasma membranes occurs primarily within the salivary glands, Diagnosis
which results in the shedding of virus that can be transmitted to
other hosts. Thus, the presence of virus in the saliva indicates Rabies should be suspected in dogs and cats evaluated for sud-
that the CNS has been infected. In some animals, death occurs den onset of behavioral changes and flaccid paralysis, especially
before the saliva becomes infected. Virus is shed by some dogs in rabies-endemic regions, or in animals imported from rabies-
for up to 13 days before the onset of clinical signs.18 endemic countries. Although a particularly high degree of suspi-
Clinical signs probably result from impaired neuronal func- cion is warranted for animals that lack a vaccination history for
tion6 and occur after an incubation period that ranges from just rabies, the disease has been reported in partially and completely
over a week to 6 months. Most dogs and cats develop disease vaccinated dogs and cats,11,17,23 so a history of rabies vaccina-
1 to 2 months after exposure. In general, the closer the bite site tion should not be used to dismiss suspicion for rabies.
is to the CNS, the shorter the incubation period. The incuba-
tion period is also influenced by the host species, age, degree of Laboratory Abnormalities
innervation of the bite site, neuroinvasiveness of the rabies virus No specific or characteristic hematological or biochemical
variant, and the amount of virus inoculated. Although 90% of changes have been reported in dogs and cats with rabies. Mild
humans develop disease within 6 months of exposure, incuba- anemia was reported in one dog.26 Analysis of the cerebrospinal
tion periods of 6 years or more have been reported.19-21 fluid (CSF) may be unremarkable or show an increase in CSF
The clinical presentations of rabies virus infection have been protein concentration and mild to marked CSF pleocytosis. Small
divided into excitatory (“furious”) and paralytic (“dumb”) lymphocytes account for most of the differential cell count.26
forms. Three phases have been described in the progression Electromyography in one dog revealed abnormalities con-
of the disease, the prodromal, furious, and paralytic phases, sistent with impaired neuromuscular transmission, including
but the stages are variable and may overlap, and signs may moderate fibrillations, positive sharp waves, and an absent
be atypical (Table 13-2). Infrequently, there is a history of a M wave.26 Changes suggestive of denervation have also been
wound, or wounds are still present at the time neurologic signs described in humans with rabies.27
136 SECTION 1  Viral Diseases
TABLE 13-3
Diagnostic Assays Available for Rabies in Dogs and Cats
Assay Specimen type Target
Direct fluorescent Brain, spinal cord, skin Rabies virus N
antibody stain- biopsies (latter not (nucleocapsid) protein
ing or immuno- recommended in
histochemistry animals), other extra­
cranial tissues
Serology Serum or cerebrospinal Antirabies virus
fluid (CSF) antibodies
Histopathology Brain, spinal cord Intracytoplasmic
eosinophilic inclusions
(Negri bodies)
RT-PCR Brain, spinal cord, saliva, Rabies virus RNA
CSF, extracranial tissues
Diagnostic Imaging
Magnetic Resonance Imaging Findings
Findings on MRI in dogs with rabies have included hyperintense
lesions on T2-weighted images, especially in the midbrain, thal-
amus, and basal ganglia, that do not enhance with contrast.28
In human patients with rabies, no significant abnormalities
may be present on MRI, or it may show hyperintensities on
T2-weighted images, especially in association with the gray mat-
ter. Lesions are frequently described in the medulla, pons, basal
ganglia, and thalamus and reflect concentrations of rabies virus
antigen.29 Contrast enhancement can be present in comatose
human patients but is usually not detected early in the course
of illness. Computed tomography commonly does not reveal
lesions in humans with rabies.
Microbiologic Testing
Diagnostic assays for rabies in dogs and cats are listed in
Table 13-3.
Direct Fluorescent Antibody Testing and Immunohistochemistry
Rabies virus diagnosis is generally based on direct fluores-
cent antibody (DFA) staining for virus on impression smears
of brain tissue obtained at necropsy. This is rapid, sensitive,
and economical and is performed only in qualified laboratories
that have been approved by local or state public health depart-
ments. In the United States, a national standardized protocol for
rabies testing is published by the Centers for Disease Control
and Prevention.30 False-negative results with DFA staining are
rare when compared with mouse inoculation testing,31 and all
animals with virus in their saliva test positive. A direct rapid
immunohistochemistry test is also available, which has a similar
sensitivity and specificity and does not require a fluorescence
microscope.32
If rabies is suspected, the animal should be handled with
extreme caution (see Prevention). Humane euthanasia should
Performance
High sensitivity and specificity on brain tissue; because
a single negative result does not rule out rabies, con-
firmatory tests such as mouse inoculation or reverse-
transcription polymerase chain reaction (RT-PCR)
are required for specimens testing negative. Sensitiv-
ity when used on extracranial tissues is relatively low.
Virus neutralization assays are the gold standard for
serology, although ELISA assays are also avail-
able. Rarely useful for diagnosis of rabies, as the
antibody response is delayed and so titers are often
negative. Titers often determined before importa-
tion of animals into rabies-free countries.
Detection of Negri bodies has low sensitivity.
Rapid (within hours), has the potential to be very
sensitive and specific; can be used to identify rabies
genotypes and strains.
be performed, and the entire head should be submitted as soon
as possible to public health authorities, so as not to delay post-
exposure prophylaxis measures for exposed individuals (see
Public Health Aspects). Opening the skull using a saw generates
aerosols and is not recommended. Although not optimal, if the
head is not available, the spinal cord can be submitted. Tissues
should be refrigerated and not frozen for optimal test sensitiv-
ity. Approved shipping containers should be used, which must
be labeled with appropriate warnings.
Antemortem, DFA testing has been used to detect rabies
virus in corneal touch impressions and frozen skin biopsy sec-
tions from the nuchal area in humans33,34 or the region of the
whiskers in dogs and cats. The sensitivity of this method is
as low as 25%, and it should not be used for clinical diag-
nosis in dogs and cats suspected to have rabies. DFA stain-
ing has also been used for detection of rabies virus antigen in
extracranial tissues at necropsy, including the tongue, salivary
glands, adrenal glands, pancreas, gastrointestinal tract, and
myocardium.35,36
Serologic Diagnosis
Serologic tests for rabies virus antibodies are often negative in
affected animals, as a result of immune evasion by the virus dur-
ing the incubation period and possibly also virus-induced immu-
nosuppression after the onset of clinical signs. If seroconversion
occurs, it is typically late in the course of illness. Prior vaccina-
tion for rabies may result in confusing results. Determination
of acute and convalescent titers on serum and determination of
CSF antibody titers can aid diagnosis of human rabies.34 In dogs
and cats, serologic testing is most commonly used to document
proof of prior vaccination, which is required for importation
into rabies-free countries. It is not suitable for determining the
need for booster vaccinations.37
Serologic methods include virus neutralization assays and
ELISA assays.38 The gold standard of rabies serology is the

rapid fluorescent focus inhibition test (RFFIT), a virus neutral-
ization assay, which assesses the ability of antibodies to prevent
viral replication in cell culture. The fluorescent antibody virus
neutralization (FAVN) test has been accepted as an alternative
for determining titers in animals before importation. A mini-
mum titer of 0.5 U/mL is required before importation to rabies-
free countries is permitted.
Virus Isolation and Mouse Inoculation
Rabies virus can be isolated in a variety of cell culture lines,
although isolation may be less sensitive than nucleic acid-based
detection methods.39 Virus isolation and inoculation of mice
with tissue from affected patients can be used to confirm rabies
virus infection by public health authorities when direct fluores-
cent antibody testing of brain tissue is negative.
Molecular Diagnosis Using the Polymerase Chain Reaction
Nucleic acid detection techniques are increasingly employed
to detect rabies virus in tissues and may ultimately replace
techniques such as isolation and histopathology.40 Reverse-­
transcriptase polymerase chain reaction (RT-PCR) assays
have been used to rapidly detect rabies virus RNA in dogs
and humans and, when used on brain tissue, are sensitive and
specific, the results correlating with those of DFA testing.41,42
RT-PCR can be used to detect virus in decomposing tissue,
when the results of other assays might be negative.43,44 Genetic
analysis of resultant PCR products can also be used to iden-
tify lyssavirus genotypes and rabies virus variants (see Table
13-1).45 For antemortem diagnosis in human patients, saliva
is more likely to test positive than CSF. A nuchal biopsy speci-
men can also be tested.46 The same appears to be true in dogs,
although again, attempts to make an antemortem diagnosis
of rabies are not recommended in animals.47 The sensitivity
of RT-PCR for detection of rabies virus in the saliva of rabid
dogs was 87%, so a negative result does not rule out a diag-
nosis of rabies.
Pathologic Findings
Gross Pathologic Findings
Gross lesions are generally absent in the CNS of dogs and cats
with rabies. Lesions consistent with a previous wound or trau-
matic event may be present elsewhere on the body.
Histopathologic Findings
Rabies virus infection results in a remarkably mild, nonsuppu-
rative polioencephalomyelitis. Changes include mild neuronal
degeneration and neuronophagia, which may be followed by
neuronal necrosis.26 Lymphoplasmacytic perivascular cuff-
ing and focal microglial hyperplasia are frequently described.
The longer the duration of illness, the more pronounced the
nonsuppurative inflammatory response. Sometimes, a spongi-
form encephalopathy with vacuolization in the gray matter is
present.16 In some animals, eosinophilic intracytoplasmic viral
inclusion bodies (Negri bodies) are identified. Negri bodies are
considered a hallmark of rabies virus infection and are most
commonly seen in tissues such as the pons, thalamus, hypo-
thalamus, and hippocampus. They are generally only seen once
neurologic signs have developed, and only in about 50% of ani-
mals that test positive using direct fluorescent antibody testing.
Inclusions that resemble Negri bodies have been described in the
brain of nonrabid cats, and so the potential for confusion and a
false positive diagnosis of rabies exists.48
CHAPTER 13  Rabies 137
Treatment and Prognosis
Immediate euthanasia and testing of dogs and cats suspected to
have rabies is recommended. Once clinical signs have developed,
rabies is almost always fatal and very few humans have sur-
vived. Most have been children, and several of those individuals
had been vaccinated for rabies before the onset of illness. In all
individuals, moderate to severe neurologic sequelae persisted. A
person in Texas appeared to survive rabies without the need for
intensive care.49 After treatment with induced coma (the “Mil-
waukee protocol”), an 8-year old girl survived rabies that may
have been acquired after a scratch from a stray cat.50 Rarely,
dogs inoculated experimentally with certain canine rabies virus
variants have recovered from rabies encephalitis, sometimes with
intermittent shedding of rabies virus in saliva after recovery.25
Immunity and Vaccination
In most cases of human rabies, no immune response is detect-
able at 7 to 10 days after the onset of clinical signs. Rabies virus
may evade immune recognition through inhibition of apoptosis
and destruction of invading T cells, which may explain the rela-
tive lack of an inflammatory response within the CNS.51
Currently available vaccines contain inactivated virus or are
canarypox-vectored recombinant rabies (cats) vaccines. Modi-
fied live rabies vaccines are no longer available in the United
States because of their association with occasional postvaccinal
rabies encephalitis in dogs and cats.52 Vaccinia poxvirus-vec-
tored glycoprotein recombinant vaccines are used for wildlife
vaccination.
All dogs and cats should be vaccinated for rabies at 3 or 4
months of age (depending on state legislation), with a booster
dose 1 year later, then every 3 years with approved inactivated
vaccines, or annually with recombinant vaccines (see Chapter
12). It has been recommended that rabies vaccines be admin-
istered as distally as possible in the right pelvic limb, to allow
better understanding of which vaccines are associated with sar-
coma formation and to permit complete removal of sarcomas by
amputation should they develop.53,54 Canine rabies vaccination
is required by law within most of the United States, and vaccines
must be administered only by (or under the direct supervision
of) a veterinarian.37 Effective vaccination of at least 70% of the
dog population is required to prevent rabies epizootics.55 Own-
ers who fail to comply with state or local requirements should
be reported to the public health authorities.
Although rare, rabies has been reported in vaccinated ani-
mals, especially when vaccinations are not up to date.23 Dogs
and cats less than 1 year of age are not considered immunized
until 28 days after the initial vaccination.37 Because of a rapid
anamnestic response, an animal is considered fully vaccinated
immediately after booster vaccination.
The use of inactivated rabies virus vaccines in cats has been
associated with formation of injection-site sarcomas.56,57 More
commonly, rabies virus vaccination is associated with pain at
the injection site, lameness, transient fever, and local cutane-
ous reactions, such as alopecia, focal cutaneous vasculitis, and
focal granulomas.58 A more generalized ischemic skin disease
characterized by variable alopecia, crusting, erosions, and ulcers
on the pinnal margins, periocular areas, tail tip, and paw pads
has been described that occurs several months after rabies virus
vaccination of dogs and begins with a lesion at the site of vac-
cination (see Chapter 12).59
138 SECTION 1  Viral Diseases

TABLE 13-4
Procedures Required by Public Health Officials for Prevention of Rabies According to the 2011 Compendium of Animal Rabies
Prevention and Control37
Situation Rabies Vaccination Procedure Rationale
History
Dog or cat exposed* Unvaccinated, dog Euthanize immediately or place in strict isolation The quarantine period is
to a rabid wild ani- or cat for 6 months. Administer rabies vaccine on based on the known incu-
mal or wild animal isolation or up to 28 days before release. bation period for rabies.
that is unavailable Not up to date Contact public health authorities, who decide pro- Animals that develop signs
for testing with booster cedures (revaccination or euthanasia) based on consistent with rabies dur-
­vaccinations biting species, local prevalence of rabies, timing ing quarantine should be
of previous vaccinations. euthanized and tested for
Vaccinated, dog or Revaccinate, and keep under owner’s control and rabies.
cat observation for 45 days.
Person bitten by a Any Confine animal and observe daily for 10 days; re- Dogs and cats shedding rabies
healthy dog or cat port illness that develops immediately to public virus within the saliva will
health authorities. If signs of rabies develop, develop illness and die
euthanize and test for rabies. Vaccination should within the 10-day quaran-
not be performed until after the isolation period tine period.
is complete.
Human bitten by a Euthanize biting animal immediately and submit
stray or unwanted for rabies testing.
dog or cat
Human bitten by a Vaccinated, human Vaccinate on day 0 and day 3 intramuscularly, in
known rabid animal the upper deltoid muscle.
Unvaccinated, Postexposure prophylaxis (injection of human
­human rabies immune globulin infiltrated at the bite
site, followed by vaccination on days 0, 3, 7, 14,
and 28)

Modified from Brown CM, Conti L, Ettestad P, et al. Compendium of animal rabies prevention and control, 2011. J Am Vet Med Assoc 2011;239:609-
617. Compendium updates are available online at http://www.nasphv.org/documentsCompendia.html.
*Exposure for rabies constitutes introduction of the virus into bite wounds, open cuts in skin, or onto mucous membranes from saliva or other po-
tentially infectious material such as neural tissue.

Oral vaccinia-vectored recombinant rabies vaccines, which
express the rabies virus glycoprotein, have been used in North
America and Europe to control the spread of rabies in wild ani-
mals such as raccoons, coyotes, and gray foxes.60 Canine and
feline rabies vaccines are not approved for use in wild animals,
which include wild animal hybrids such as wolf hybrids and
Savannah cats. Wolf hybrids and Savannah cats are considered
unvaccinated when decisions must be made regarding quaran-
tine and euthanasia (Table 13-4).
Prevention
Prevention of rabies is dependent on widespread education of the
public regarding the need to vaccinate dogs and cats for rabies,
and the risks of wildlife contact. Control of stray dogs and cats
and wild animal vaccination programs also help to prevent the
disease. Ownership and importation of wild animals and hybrids
is discouraged. Puppies and kittens that have not yet reached the
age for rabies vaccination should be kept away from wildlife.
According to U.S. regulations, unvaccinated dogs and cats
that have been exposed to a rabid animal should be euthanized
immediately or vaccinated and placed in strict isolation, without
direct contact with people or other animals, for 6 months.37 If
the biting animal is a wild animal that is unavailable for test-
ing, the biting animal is presumed to be rabid. Animals that are
overdue for a booster vaccination are evaluated on a case-by-case
basis by public health authorities. Dogs and cats that are cur-
rently vaccinated should be revaccinated immediately (within 48
hours), kept under the owner’s control, and observed for 45 days.
Any illness in isolated or confined animals should be reported to
local public health officials. If clinical signs suggestive of rabies
develop, euthanasia and rabies testing should be performed.
Animals suspected to have rabies should be isolated from
other animals and humans and handled with extreme caution,
using heavy gloves, catch poles, and cages for restraint. Care
should be taken not to allow people’s skin to come into con-
tact with salivary secretions. Signs that warn of the possibility
of rabies should be placed on the cages of animals that are
hospitalized for any period of time. If the risk of rabies is low,
the animal should be confined and observed for 10 days. The
clinician in charge should keep a list of all in-contact person-
nel during that period, and procedures should be minimized.
High-risk rabies suspects should be confined in isolation using
an established hospital protocol (see Chapter 11). Marked
improvement in an animal’s condition that occurs during a
10-day period is not consistent with a diagnosis of rabies. All

specimens submitted to the laboratory should be labeled with
warning labels.
Public Health Aspects
Each year, 50,000 to 55,000 people die from rabies worldwide.
Approximately half those numbers occur in India alone, and it
has been estimated that 3 billion people continue to be at risk
of rabies virus infection in more than 100 countries.61 A large
proportion of these people are children who have been attacked
by rabid dogs. In the United States, human deaths due to rabies
have decreased from more than 100 per year in the early 20th
century to 2 to 3 per year.62 Most humans in the United States
are infected as a result of contact with insectivorous bats, which
often appear injured or are behaving abnormally (including fly-
ing during the day), and, depending on the bat species, around
5% to 40% of dead or ill bats submitted for testing have
rabies.63,64 Seemingly insignificant contact with tiny bats, par-
ticularly the eastern pipistrelle and silver-haired bats, is reported
in many human rabies cases (cryptic bat rabies). Despite the
high prevalence of raccoon rabies in the eastern United States,
the only human death associated with a raccoon rabies variant
was reported in 2003.65
Prodromal signs of rabies in humans include malaise, fever,
and pain, itching, or paresthesia at the site of the bite. People
with furious rabies become nervous and hyperexcitable, after
which signs of hydrophobia, aerophobia, confusion, and
aggression develop. Autonomic signs such as hypersalivation,
vomiting, miosis or mydriasis, excessive sweating, and priapism
may occur. Paralytic rabies can be confused with Guillain-Barré
syndrome.34
Sick domestic and wild animals that bite humans in regions
where rabies is endemic and that subsequently die should be
tested for rabies without delay. Prompt and aggressive treat-
ment of wounds including irrigation under pressure with 20%
aqueous soap solution, and application of a greater than 50%
ethanol solution or povidone-iodine, can dramatically reduce
the risk of contracting rabies after a bite.
The chain of events that must occur after a dog or cat bites
a human are listed in Table 13-4 and depend on whether the
animal is owned or stray and the animal’s vaccination status.37
Public health authorities must be notified when potential expo-
sure occurs and make the final decision as to how these animals
are handled.
Postexposure prophylaxis (PEP) is administered to humans
after a bite from a known or suspected rabid animal. In the
United States it consists of an injection of human rabies immune
globulin (H-RIG), ideally with at least half of the whole dose
in the region of a bite site as soon as possible within the first 7
days of the bite, followed by vaccination by the intramuscular
route on days 0, 3, 7, 14, and 28 in the upper deltoid muscle.
Fewer than 5 humans develop rabies each year in the United
States, but PEP is given to 20,000 to 40,000 people each year,
with on average 50 humans receiving PEP for each rabies case
investigated.
Depending on risk, certain individuals may receive prophy-
lactic vaccines against rabies. These individuals include vet-
erinarians, veterinary students and staff, dog catchers, rabies
researchers, rabies diagnosticians, wildlife workers, and travel-
ers visiting rabies-enzootic regions. After exposure to a rabid
animal, administration of immunoglobulin to these individu-
als is not necessary. Instead, rabies vaccine boosters are given
CHAPTER 13  Rabies 139
immediately and 3 days later. The major vaccine in use in
the United States is the human diploid cell vaccine (HDCV),
although in developing countries, access to this vaccine is
limited. Among animal workers, antibody titers are generally
checked every 2 years, with administration of a booster dose if
necessary.
SUGGESTED READINGS
Dietzschold B, Li J, Faber M, et  al. Concepts in the pathogenesis of
rabies. Future Virol. 2008;3:481-490.
Lackay SN, Kuang Y, Fu ZF. Rabies in small animals. Vet Clin North
Am Small Anim Pract. 2008;38:851-861:ix.
Public health response to a rabid kitten-four states, 2007. MMWR
Morb Mortal Wkly Rep. 2008;56(51-52):1337-1340.
Recovery of a patient from clinical rabies–California, 2011. MMWR
Morb Mortal Wkly Rep. 2012;61:61-65.
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CHAPTER 14
Canine Parvovirus Infections and
Other Viral Enteritides
Jane E. Sykes
Overview of Canine Parvoviral Enteritis
First Described: 1978, worldwide, Appel and others1
Cause: Canine parvovirus-2 variants (Family Parvoviridae,
subfamily Parvovirinae, Genus Parvovirus)
Affected Hosts: Dogs and other Canidae such as coyotes,
foxes, and wolves; cats
Geographic Distribution: Worldwide
Route of Transmission: Direct contact with virus in feces and
vomitus as well as contact with contaminated fomites
Major Clinical Signs: Fever, lethargy, inappetence, vomiting,
diarrhea, dehydration. Sudden death or tachypnea due to
myocarditis occurs rarely.
Differential Diagnoses: Canine distemper virus infection,
other canine viral enteritides, dietary indiscretion, toxins,
gastrointestinal foreign body, enteric parasitic infections
such as giardiasis and helminth infections, enteric bacte-
rial infections such as salmonellosis, salmon poisoning
disease, pancreatitis, hypoadrenocorticism, inflammatory
bowel disease
Human Health Significance: CPV-2 variants do not infect
humans
Etiology and Epidemiology
Canine parvovirus is the most widely recognized cause of trans-
missible viral diarrhea in dogs and one of the most common
infectious diseases of dogs worldwide. It is caused by variants of
canine parvovirus-2 (CPV-2), which are members of the genus
Parvovirus. CPV-2 emerged in the early to mid-1970s and
caused a worldwide pandemic of illness in dogs.2 The spread of
the virus worldwide occurred over a remarkable period of about
6 months. CPV-2 may have been derived from feline panleuko-
penia virus (FPV) or a closely related virus of wild carnivores.
It has since mutated to CPV-2a in 1979, CPV-2b in 1984, and,
most recently, CPV-2c, which was first detected in Italy in 2000
and has subsequently been found worldwide, with the exception
of Australia. Separate lineages have been identified in different
geographic locations worldwide.3 The virus uses the transferrin
receptor to enter host cells. With the mutation from CPV-2 to
CPV-2a, the virus developed the ability to replicate readily in
feline cells, and CPV-2a, CPV-2b, and CPV-2c are now respon-
sible for some cases of feline viral enteritis.4,5 Feline parvoviral
enteritis is further discussed in Chapter 19. CPV-2 variants must
be differentiated from CPV-1, also known as canine minute
virus, which belongs to the genus Bocavirus. In general, CPV-1
is thought to have minimal pathogenic potential, but it has been
associated with abortion in pregnant dogs; respiratory, cardiac,
and gastrointestinal signs in neonatal dogs; severe gastroenteri-
tis in adult dogs; and possibly neurologic signs in dogs.6-8
Parvoviruses are small, non-enveloped single-stranded DNA
viruses (Figure 14-1, A) that can survive for long periods (over
1 year) in the environment. As a result, contact with virus that
persists in the environment is an important means of transmis-
sion. Insects and rodents may also serve as mechanical vectors
for the virus. Canine parvovirus requires the presence of mitoti-
cally active cells in order to replicate. Young animals (6 weeks
to 6 months, and especially those less than 12 weeks of age) are
more likely to develop severe illness; however, disease can also
occur in unvaccinated or improperly vaccinated adult dogs. In
North America, Rottweilers, American pit bull terriers, Dober-
man pinschers, English springer spaniels, and German shep-
herd dogs appear to be at increased risk for development of
parvoviral enteritis,9,10 but this has not been the case in other
geographic locations. A seasonal distribution of disease has
been reported in some geographic locations, which may reflect
times when dogs access the outdoors and contact virus in the
environment. For example, in Saskatoon, Canada, dogs were
three times more likely to be admitted with parvoviral enteritis
in July, August, and September, compared with the rest of the
year.9 In other locations, the seasonal pattern of disease has dif-
fered or been nonexistent.11,12 For dogs older than 6 months
of age, intact males were twice as likely as females to develop
parvoviral enteritis.9 A surveillance study from Australia found
a correlation between clusters of parvoviral enteritis and regions
of relative socioeconomic disadvantage.12
Other viral pathogens that have been associated with enteri-
tis and diarrhea in dogs are canine distemper virus (CDV)
(see Chapter 15), canine enteric coronavirus (Figure 14-1, B),
rotaviruses, astroviruses, adenoviruses, caliciviruses, and
novel viruses that include a norovirus, kobuvirus, sapovirus,
and possibly also a circovirus.13-17 Canine enteric coronavirus
primarily causes mild diarrhea in puppies that are less than
6 weeks of age and may be found in co-infections with other
viral causes of gastroenteritis, including CPV-2 variants. Rarely,
it has been identified as a more significant cause of diarrhea in
young dogs.18 This chapter focuses on canine parvoviral enteri-
tis, which is the most widely recognized and pathogenic viral
enteritis of dogs.
Clinical Features
Signs and Their Pathogenesis
Transmission of parvovirus and other viral causes of gastroen-
teritis occurs by the fecal-oral route, after exposure to virus in
feces or vomit, or importantly, virus that persists on fomites.
141
142 SECTION 1  Viral Diseases
Spike glycoprotein
Helical
nucleocapsid
A B
FIGURE 14-1  A, General structure of canine parvovirus. The virus is non-enveloped,
is 25 nm in diameter, and has icosahedral symmetry. B, Structure of a coronavirus, which
is an enveloped virus. Canine parvovirus is about one-quarter the size of a coronavirus.
Virus is shed for a few days before the onset of clinical signs,
and shedding declines considerably after 7 days.19 The severity
of clinical signs depends on factors such as virus strain and host
immunity, which is affected by stressors such as weaning and
overcrowding, maternal antibody, and the presence of concur-
rent infections such as other enteric viral and parasitic infec-
tions. Subclinical infections are probably widespread.
The incubation period for canine parvoviral enteritis is 7 to
14 days in the field, but shorter incubation periods (as short as 4
days) have been observed with experimental infections. The virus
replicates in oropharyngeal lymphoid tissues, after which viremia
occurs. Damage occurs to rapidly dividing cells in the gastroin-
testinal tract, thymus, lymph nodes, and bone marrow. Affected
gastrointestinal tissues include the epithelium of the tongue, oral
cavity, esophagus, and intestinal tract, and especially the germi-
nal epithelial cells of the intestinal crypts. Neutropenia results
not only from infection of the marrow but also sequestration of
neutrophils in damaged gastrointestinal tissue. Malabsorption
and increased intestinal permeability result. Secondary bacterial
infections of the gastrointestinal tract, which may be followed
by bacterial translocation, bacteremia, and endotoxemia, play
a key role in the pathogenesis of the disease. Mucosal candidia-
sis has also been described in puppies with parvoviral enteritis
(see Chapter 67). The outcome is clinical signs of fever, leth-
argy, inappetence, vomiting, diarrhea, rapid dehydration, and
abdominal pain. Diarrhea is often liquid, foul-smelling, and may
contain streaks of blood or frank blood. Ascarid nematodes may
be identified in the vomitus of some dogs. Dogs with canine par-
voviral enteritis have evidence of disordered coagulation, with
decreased antithrombin activities, prolonged activated partial
thromboplastin time, increased thromboelastography maximum
amplitude, and increased fibrinogen concentrations. Catheter
and organ thrombosis may occur.20 Secondary bacteremia may
be associated with multiple organ failure and death.
Infection of the dam by CPV-2 variants early in gestation
can lead to infertility, resorption, or abortion. Puppies that are
infected in utero or up to 2 weeks of age may develop viral
myocarditis, which results in signs of sudden death or conges-
tive heart failure.21-23 Damage to the developing myocardium
usually occurs up to the first 2 weeks of life, but clinical signs of
myocardial damage may be delayed until up to 2 months of age.
Cerebellar hypoplasia has been rarely reported in dogs after in
utero infection24 but is more common in kittens infected with
FPV (see Chapter 19). Generalized infection has been reported
in neonatal puppies, with hemorrhage and necrosis within the
brain, liver, lungs, kidneys, lymphoid tissues, and gastrointesti-
nal tract.25 Because maternal antibody protects puppies during
FIGURE 14-2  Target sites of replication of selected enteric viral pathogens. The most
pathogenic enteric viruses, such as canine parvovirus, replicate and destroy crypt epithelial
cells.
this period, the incidence of neonatal complications of parvo-
virus infection and myocarditis has declined dramatically since
the virus first emerged, because of widespread vaccination and
exposure of adult animals.
Neurologic signs in puppies with parvoviral enteritis may
result from hypoxia secondary to myocarditis, hypoglycemia,
or intracranial thrombosis or hemorrhage. The possibility of
co-infection with CDV should also be considered. The DNA
of CPV-2 variants has also been detected in the central nervous
system,26 and there have been rare reports of leukoencephalo-
malacia in association with infection.27,28
In contrast to canine CPV-2 variants, the replication of less
pathogenic enteric viruses is restricted to the intestinal tract,
and the crypt epithelial cells are generally spared (Figure 14-2).
Canine enteric coronavirus, for example, infects the mature
enterocytes at the tips of the villi. Crypt cell hyperplasia occurs
to replace the damaged cells. The villi become shortened or dis-
torted, which leads to malabsorption and diarrhea.
Physical Examination Findings
Physical examination of puppies with parvoviral enteritis often
reveals fever (up to 41°C or 105°F), lethargy, weakness, dehy-
dration, and abdominal tenderness and a fluid-filled intestinal
tract on palpation (Figure 14-3). Vomiting or diarrhea may
occur during the examination, or there may be evidence of diar-
rhea or frank blood on the perineum or the rectal thermometer.
Occasionally abdominal palpation reveals a tubular mass as a
result of intestinal intussusception. Ulcerative glossitis can occur
in some puppies. Mucosal pallor, prolonged capillary refill time,
or rarely hypothermia may be observed in some dogs.29 Septic
shock may be associated with tachycardia or bradycardia, men-
tal obtundation, and poor pulse quality. Uncommonly, neuro-
logic signs such as tremors and seizures are observed. Puppies
with myocarditis may be tachypneic and have increased lung
sounds as a result of congestive heart failure. Erythema multi-
forme has been reported in dogs with canine parvoviral enteritis;
these dogs had generalized cutaneous and mucosal ulceration as
well as swelling of the pinnae and paws.30,31
 CHAPTER 14  Canine Parvovirus Infections and Other Viral Enteritides 143

Diagnosis
Laboratory Abnormalities
Complete Blood Count
The most common abnormalities found on the CBC are leuko-
penia, neutropenia, and lymphopenia (Table 14-1). Toxic neu-
trophils and monocytopenia may also be present. Only around
one third of dogs had leukopenia in one study.29 Leukopenia
can develop after the onset of gastrointestinal signs, when
puppies are first brought for examination. Some dogs have
leukocytosis due to a neutrophilia and monocytosis. Although
the presence of leukopenia supports a diagnosis of parvoviral
enteritis, other severe gastrointestinal infections such as sal-
monellosis can also cause leukopenia and diarrhea, so it is not
specific for diagnosis of the disease. Thrombocytosis or, less
commonly, thrombocytopenia may also occur.29 Some pup-
pies develop anemia as a result of gastrointestinal blood loss,
which may be non regenerative or become regenerative.

Serum Biochemical Tests

The serum biochemistry panel in dogs with parvoviral enteritis
often shows hypoproteinemia, hypoalbuminemia, and hypogly-
cemia. Mild hyperglycemia has also been reported. Electrolyte
abnormalities such as hyponatremia, hypochloremia, and hypo-
kalemia may occur (Table 14-2). Occasionally severe dehydration
results in prerenal azotemia. Puppies with bacterial sepsis may
develop increased liver enzyme activities and hyperbilirubinemia.

FIGURE 14-3  Obtundation in a 3-month-old intact male standard poodle puppy
with canine parvovirus enteritis. The dog had been vaccinated for canine parvovirus at 8
and 10 weeks of age.
Coagulation Profile
Coagulation abnormalities have been reported in a small num-
ber of dogs with parvoviral enteritis. When abnormalities occur,
findings include prolonged activated partial thromboplastin
time, decreased antithrombin activity, increased fibrinogen

TABLE 14-1
Complete Blood Count Findings at Admission in 45 Dogs Diagnosed with Canine Parvoviral Enteritis at the UC Davis VMTH
Percent below Percent within Percent above Range for
Reference the Reference the Reference the Reference Dogs with CPV Number
Test Range Range Range Range Enteritis Tested
Hematocrit (%) 40-55 71 29 0 21-53 45
MCV (fL) 65-75 29 71 0 54-74 45
MCHC (g/dL) 33-36 24 69 7 30-37 45
Neutrophils (cells/µL) 3000-10,500 56 31 13 8-22,453 45
Band neutrophils 0-rare 0 31 69 0-1582 45
(cells/µL)
Monocytes (cells/µL) 150-1200 20 60 20 11-2475 45
Lymphocytes (cells/µL) 1000-4000 49 22 0 165-3698 45
Eosinophils (cells/µL) 0-1500 0 100 0 0-1236 45
Platelets (cells/µL) 150,000-400,000 5 60 35 103,000- 43
639,000

Note: Adult reference ranges were used by the laboratory.

CPV, Canine parvovirus.
144 SECTION 1  Viral Diseases

TABLE 14-2
Findings on Serum Biochemistry Analysis in 27 Dogs with Canine Parvoviral Enteritis at the UC Davis VMTH
Percent below Percent within Percent above Range for Dogs
Reference the Reference the Reference the Reference with Parvoviral Number of
Test Range Range Range Range Enteritis Dogs Tested
Sodium (mmol/L) 145-154 85 15 0 132-147 26
Potassium (mmol/L) 3.6-5.3 4 92 4 3.4-5.9 26
Chloride (mmol/L) 108-118 65 31 4 94-120 26
Bicarbonate (mmol/L) 16-26 8 92 0 14-25 26
Calcium (mg/dL) 9.7-11.5 30 70 0 7.4-11.5 27
Phosphorus (mg/dL) 3.0-6.2 0 52 48 3.1-12.3 27
Creatinine (mg/dL) 0.3-1.2 37 59 4 <0.2-1.6 27
BUN (mg/dL) 5-21 0 85 15 5-48 27
Albumin (g/dL) 3.0-4.4 70 30 0 0.9-3.8 27
Globulin (g/dL) 1.8-3.9 26 67 7 1.4-4.5 27
Cholesterol (mg/dL) 135-361 4 89 8 108-460 26
Total bilirubin (mg/dL) 0-0.2 0 89 12 0-1.7 26
ALT (U/L) 19-67 15 62 23 10-165 26
ALP (U/L) 21-170 0 42 58 56-397 26

Note: Adult reference ranges were used by the laboratory.

concentrations, and increased thromboelastography maximum
amplitude.20 An increase in D-dimers or fibrin degradation
products has not been reported. More research is warranted
to understand the range of coagulation abnormalities that can
occur in parvoviral enteritis.
Diagnostic Imaging
Plain Radiography
Plain abdominal radiography in dogs with parvoviral enteritis
may show poor serosal detail (often due to lack of intra-abdom-
inal fat in puppies) and a fluid- and gas-filled gastrointestinal
tract. Abdominal radiography is usually performed to assess for
the presence of a gastrointestinal foreign body.
Sonographic Findings
Findings on abdominal ultrasonography in canine parvoviral
enteritis are nonspecific but can include a thickened gastroin-
testinal mucosa, mild peritoneal effusion, fluid distention of the
gastrointestinal tract, and decreased gastrointestinal motility.
Mild mesenteric lymphadenopathy may be present. Abdominal
ultrasound is useful to confirm a diagnosis of secondary intesti-
nal intussusception.
Microbiologic Tests
Diagnostic assays for canine parvoviral enteritis in dogs are
listed in Table 14-3.
Fecal Parvovirus Antigen ELISA
The most widely used assay for diagnosis of canine parvoviral
enteritis is an in-house fecal antigen ELISA, which is performed
on a rectal swab specimen. Several assays are available, and
although they detect all variants of CPV-2 including CPV-2c,
their sensitivities and specificities vary.32,33 Sensitivity is par-
ticularly problematic, because viral shedding is transient, and
antibody present may bind viral antigen so that it is unavail-
able for reaction with the assay. The sensitivities of three com-
mercially available fecal antigen assays (SNAP Parvo antigen,
IDEXX Laboratories GmbH; FASTest Parvo Strip, Scil Ani-
mal Care Company GmbH; Witness Parvo Card, Selectavet
GmbH) were 50%, 40%, and 60%, respectively, when com-
pared to immunoelectron microscopy, and 18%, 16%, and
26%, respectively, when compared with results of a fecal PCR
assay.33 Because some dogs that lack evidence of gastrointes-
tinal signs can be positive using PCR assay, immunoelectron
microscopy may be a more appropriate gold standard for dis-
ease (although it is not widely available for routine diagno-
sis). In another study, the sensitivity of a fecal antigen test for
detection of CPV-2a, CPV-2b, and CPV-2c, on specimens that
contained high CPV DNA loads (>105 copies/mg of feces as
determined with real-time PCR) was 80%, 78%, and 77%,
respectively.32
When compared with PCR and immunoelectron microscopy
of stool, the specificities of the three commercially available
antigen assays above were 98%, 98%, and 92%, respectively,33
so false positives were uncommon. It has been suggested that
false-positive antigen tests can occur 4 to 8 days after vacci-
nation with attenuated live CPV-2 vaccines.34 In kittens, false-
positive test results did occur after vaccination for FPV, but
they were more likely to occur with some assays as opposed to
others, were generally weak positives, and occurred even after
vaccination with inactivated vaccines.35 Similar studies have not
been reported for CPV.
 CHAPTER 14  Canine Parvovirus Infections and Other Viral Enteritides 145

TABLE 14-3
Diagnostic Assays Available for Canine Parvoviral Enteritis in Dogs
Assay Specimen type Target Performance
Fecal antigen Feces CPV antigen Specificity for detection of virus nears 100%, but weak
ELISA false positives have the potential to occur after immu-
nization with attenuated live vaccines. False negatives
are common (low sensitivity).
Hemagglutina- Feces CPV antigen Inexpensive and rapid. Sensitivity and specificity in natu-
tion assay rally infected dogs has not been well established.
Histopathology Usually necropsy Crypt necrosis with Can be used for diagnosis at necropsy. In situ hybridiza-
specimens, especial- intranuclear inclusions; tion may be most sensitive for detection of virus in
ly gastrointestinal parvovirus antigen tissues.
tissues with IHC or parvovi-
rus DNA with in situ
hybridization
Polymerase chain Feces, tissue species CPV DNA Sensitivity and specificity may vary depending on assay
reaction (PCR) design. Attenuated live vaccine virus may be detected
in feces for days to weeks after vaccination, but some
assays differentiate between vaccine and field virus.
Because of the high sensitivity of some assays, the
significance of a positive result may be difficult to
interpret. False negative results may occur as a result
of inhibition of PCR by components of feces. Degrada-
tion of nucleic acid during specimen transport is more
problematic for RNA viruses such as canine coronavi-
rus.
Fecal electron Feces Virus particles Not widely available, turnaround time can be slow,
microscopy and may be expensive. Requires the presence of large
amounts of virus.
Virus isolation Feces, tissues CPV Difficult, not widely available. Used as a research tool.

CPV, Canine parvovirus (refers to CPV-2 variants); IHC, immunohistochemistry.

Hemagglutination Testing
Canine parvovirus agglutinates erythrocytes, and so the presence
of the virus in stool can be detected with a simple hemagglutina-
tion test that involves mixing a suspension of feces with porcine
erythrocytes. Agglutination of erythrocytes in a microwell plate
or on a slide indicates the presence of parvovirus in the feces.36
The sensitivity and specificity of these assays in dogs with and
without natural parvoviral infections in the field require further
investigation.
Molecular Diagnosis Using the Polymerase Chain Reaction
Several commercial veterinary diagnostic laboratories now offer
real-time PCR assays for detection of CPV-2 variants and other
enteric viral pathogens (such as canine coronavirus). Assays
may detect as few as 1000 copies of viral DNA per milligram of
stool. Turnaround times are less than 24 hours in some labora-
tories. PCR assays are useful when fecal antigen tests are nega-
tive but parvoviral enteritis is still suspected as a diagnosis, or
when canine enteric coronavirus infection is a potential cause of
illness (because PCR panels that assay for parvovirus DNA also
often include an assay for canine enteric coronavirus RNA).
Unfortunately, although infrequent, positive PCR assay results
for CPV can occur in dogs without signs of gastroenteritis or in
dogs with chronic diarrhea, and so it may be difficult to ascer-
tain whether a positive parvovirus PCR result indicates that
CPV is the cause of a dog’s illness. Attenuated live vaccine virus
can also be detected in the feces with PCR assays after vaccina-
tion, although assays have been designed that can differentiate
between vaccine and wild-type virus.37 It is not yet clear for how
long after vaccination false-positive PCR results might occur,
and this could vary based on the design of the PCR assay used.
Both vaccine and field virus have been detected simultaneously
in some dogs using these assays. In the future, quantitation of
virus loads in feces using real-time PCR may be helpful for inter-
pretation of the significance of a positive PCR assay result.
Fecal Electron Microscopy
Fecal electron microscopy is still offered by some institutions
for diagnosis of viral enteritis. It is generally used on a research
basis when viral enteritis is suspected but a diagnosis cannot be
made with antigen tests or PCR assays. Fecal electron micros-
copy may facilitate diagnosis of other viral infections such as
rotavirus, norovirus, and coronavirus infections. Turnaround
time may be slow. Generally speaking, large amounts of virus
must be present for results to be positive, and technical expertise
is required to accurately identify virus in the stool.
146 SECTION 1  Viral Diseases
FIGURE 14-4  Discoloration of the small intestinal wall and serosal hemorrhage in
a puppy that died of canine parvoviral enteritis. (Courtesy University of California, Davis
Veterinary Anatomic Pathology Service.)
Virus Isolation
Canine parvovirus variants can be isolated in canine and feline
cells, but isolation is difficult, and the virus shows minimal
cytopathic effects. As a result, virus isolation is rarely used for
diagnosis and is not widely available. It remains important as a
research tool.38
Serology
Antibodies to CPV-2 can be measured in the laboratory using
hemagglutination inhibition (see Chapter 2). In addition, an
in-clinic ELISA assay is available for semiquantitative measure-
ment of antibodies to CPV-2. These assays are generally used
to assess the need for vaccination, rather than for diagnosis of
CPV-2 enteritis, because affected dogs are either seronegative
or previous vaccination or material antibodies confound early
serodiagnosis.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with CPV enteritis include
thickening and discoloration of the intestinal wall with serosal
hemorrhage (Figure 14-4) and enlarged, edematous abdominal
lymph nodes. The intestine may contain bloody liquid con-
tents, and mucosal hemorrhage may be identified. Pale areas
may be seen within the myocardium of dogs with parvoviral
myocarditis.
Histopathologic Findings
The major histopathologic finding is necrosis of the crypt epi-
thelium in the small intestine, with widespread systemic lym-
phoid depletion and necrosis. The crypts can be dilated and
distended with cellular debris and mucus (Figure 14-5). Pro-
liferation of crypt enterocytes may be observed as part of the
recovery response. Intestinal villi are collapsed, shortened, and
fused, with attenuation of the epithelial lining, and there may be
mild to severe fibrinous inflammation and hemorrhage. Myeloid
depletion may be found in the bone marrow. Parvoviral myocar-
ditis is characterized by myocardial degeneration and necrosis,
with a lymphocytic inflammatory infiltrate. Myocardial fibrosis
can also be present. Rarely, central nervous system lesions con-
sisting of leukoencephalomalacia have been described.27 Viral
intranuclear inclusions may be visible in some cells, especially
the intestinal crypt epithelium. Immunohistochemistry can be
used to detect viral antigen in the gastrointestinal tract, mar-
row, lymphoid tissues, and rarely in the myocardium. In situ
hybridization (see Chapter 5) can also be used to detect virus in
histopathology specimens and may have greater sensitivity than
immunohistochemistry.39,40
Treatment and Prognosis
Antimicrobial Treatment and Supportive Care
Treatment of CPV enteritis involves supportive care and treat-
ment of secondary bacterial infections with antimicrobial drugs
(Table 14-4). Whenever possible, the patient should be hospi-
talized in isolation. Appropriate fluid therapy and maintenance
of adequate blood glucose concentrations are the most critical
aspect of treatment. Whenever possible, fluids should be given
intravenously and supplemented as needed with potassium
chloride and dextrose. Blood glucose concentrations should
be monitored at least twice daily, and more frequent monitor-
ing may be indicated if hypoglycemia is present. Although not
generally recommended for puppies that are vomiting or dehy-
drated, administration of subcutaneous fluids and antimicrobial
drugs in the home can sometimes result in recovery when client
finances do not permit treatment in hospital. Fluids adminis-
tered subcutaneously should never be supplemented with dex-
trose, because dextrose is hyperosmotic and can cause further
dehydration, as well as injection-site reactions. Unfortunately,
the owners of many dogs with parvoviral enteritis lack finan-
cial resources for treatment. In these situations, the inability to
afford vaccination may have been a reason the puppy develops
parvoviral enteritis in the first place.
An antimicrobial drug or drug combination with activity
against gram-negative and anaerobic bacteria should be admin-
istered parenterally. Injectable ampicillin or cefazolin alone may
be sufficient for many dogs, but puppies that have hemorrhagic
diarrhea or evidence of the systemic inflammatory response syn-
drome (SIRS) should probably be treated with a combination of
a penicillin and a fluoroquinolone, or a combination of a peni-
cillin and an aminoglycoside. Use of fluoroquinolones in young,
rapidly growing animals has been associated with cartilage
damage (see Chapter 8), but when used for the short periods of
time required to treat parvoviral enteritis, this may not be of sig-
nificant concern. Proper hydration is critical before aminogly-
cosides are used because of their potential for nephrotoxicity.
Treatment with antiemetics (such as a constant rate infusion
of metoclopramide or parenteral ondansetron), H2 blockers
such as famotidine, whole blood or plasma transfusions, col-
loids such as hetastarch, or partial or total parenteral nutrition
may be indicated in some dogs. Placement of a central line or
multilumen catheter may be necessary in severely ill puppies,
but strict sterile technique must be adhered to because of the
potential for hospital-associated infection. In general, unless
absolutely necessary, invasive surgical procedures and the use
of parenteral nutrition solutions should be avoided in puppies
with severe neutropenia. Whether plasma or hetastarch is the
treatment of choice for dogs with low colloid oncotic pres-
sure requires further study. Plasma may offer benefit over het-
astarch in that it contains antibodies from immune dogs, but
titers may not be sufficient to be beneficial, and most puppies
 CHAPTER 14  Canine Parvovirus Infections and Other Viral Enteritides 147

A B
FIGURE 14-5  A, Segmental crypt necrosis in the ileum of a dog infected with a CPV-2 variant. There is severe loss of crypt epithelial cells. Hematoxylin and eosin (H&E) stain.
B, Jejunum of a dog after infection by a CPV-2 variant. Regenerating epithelial cells, which here are nested in an inflamed jejunal lamina, have a large and bizarre appearance and resemble
adenoma cells. As a result, the disease has been termed adenomatosis. Parvovirus antigen was no longer detectable by immunohistochemistry at this stage. The crypts in the bottom left
corner have a more normal appearance. H&E stain. (Courtesy Dr. Patricia Pesavento, University of California, Davis Veterinary Anatomic Pathology Service.)

been associated with acute kidney injury in critically ill human

TABLE 14-4 patients.41 Early enteral nutrition with a nasogastric feeding
Medications That May Be Used in Conjunction with Fluid tube was associated with reduced hospitalization times in one
study, as compared to withholding food until vomiting had
Therapy to Treat Canine Parvoviral Enteritis
ceased (Figure 14-6).42 Gastric suction should be performed
through the tube before food is administered.
Interval
Other treatments that have been investigated include anti-
Drug Dose (mg/kg) Route (hours)
endotoxin sera, recombinant bactericidal permeability-increasing
Ampicillin sodium 20 IV 6 (BPI) protein, recombinant granulocyte colony-stimulating
Cefazolin sodium 20 IV 8 factor (G-CSF), recombinant feline interferon-omega (rfIFN-ω,
Enrofloxacin* 5 IV 24 Virbagen omega), and oseltamivir. Anti-endotoxin sera, recom-
binant BPI protein, and human recombinant G-CSF were
Ondansetron 0.5 to 1 IV 12 not found to be beneficial. Although neutrophil counts were
Maropitant citrate 1 SC 24 higher and hospital times were shorter in dogs treated with
Metoclopramide 1-2 mg/kg/d IV CRI recombinant canine G-CSF, survival times in these dogs were
decreased.43 Treatment of parvovirus infections with G-CSF
Famotidine 0.5 IV 12 to 24
might cause harm, because the increased cell turnover induced
CRI, Constant-rate infusion. by the drug might promote parvovirus replication. Treatment of
*Has been associated with cartilage injury in growing animals. Pro- puppies with parvoviral enteritis with rfIFN-ω in a number of
longed use (>7 days) is not recommended. See Chapter 8. placebo-controlled trials has been associated with reduced dis-
ease severity and, in some studies, significantly reduced mortal-
ity, but it has not been beneficial for treatment of FPV infections
show evidence of an antibody response within 3 days after onset (see Chapter 19).
of clinical signs. Hetastarch has anticoagulant properties that Oseltamivir inhibits the neuraminidase of influenza viruses
could be beneficial in light of the hypercoagulable state that has and does not have specific anti-parvoviral activity, but it has
been documented in canine parvoviral enteritis, but these effects been widely used for treatment of canine parvoviral enteritis. As
also have the potential to increase mortality, and hetastarch has canine parvovirus does not possess a neuraminidase, it has been
148 SECTION 1  Viral Diseases
FIGURE 14-6  Puppy in Figure 14-2 after placement of a nasogastric feeding tube.
hypothesized that oseltamivir instead may act on the neuramini-
dases of bacteria that are normally responsible for secondary
bacterial infections in parvovirus enteritis, primarily those of the
gastrointestinal tract. A single prospective, randomized, masked,
placebo-controlled trial of 35 dogs with parvovirus enteritis
showed that dogs treated with oseltamivir (2 mg/kg PO q12h)
had no significant drop in their leukocyte count, whereas
untreated dogs had a significant drop in their leukocyte count
in the first 5 days of hospitalization.44 Treated dogs also
gained weight during hospitalization, whereas untreated dogs
lost weight. However, there was no difference in hospitaliza-
tion time, clinical scores, morbidity, or mortality between the
two groups, and the number of dogs in each group was small.
The authors acknowledged the potential concerns that relate to
administration of an oral medication to dogs with enteritis, with
possible variability in drug absorption. A major concern that
relates to treatment of canine parvoviral enteritis with oselta-
mivir is the possibility of selection for resistant mutants among
influenza viruses if widespread use of the drug occurs in veteri-
nary clinics. Given the restrictions on the use of this drug for
treatment of human influenza virus infections, further investiga-
tion is required before the widespread use of oseltamivir can be
recommended for treatment of CPV enteritis. More informa-
tion on canine G-CSF, rfIFN-ω, and oseltamivir can be found in
Chapter 7 of this book.
After recovery from viral enteritis, intestinal parasites
should be treated with a broad-spectrum anthelmintic such as
fenbendazole.
Prognosis
The prognosis for viral enteritis in puppies varies with the severity
of illness and the owners’ ability to afford appropriate treatment.
Survival rates for puppies with CPV-2 enteritis have ranged from
9% in untreated puppies to greater than 90% with aggressive
treatment in tertiary referral hospitals.10,45-47 Factors that have
been related to mortality include the presence of initial leukope-
nia or lymphopenia, monocytopenia, neutropenia, and evidence
of SIRS.29,48,49 SIRS was defined as the presence of at least three of
the following four criteria: heart rate greater than 140 beats/min,
temperature above 102.5°F or below 100°F, and white blood
cell counts greater than 17,000 or less than 6000 cells/µL (see
Chapter 86). Survival may also be lower in Rottweilers when
compared to other breeds and in young puppies (less than 7 to
12 weeks of age).50 In Australia, euthanasia was more likely to
occur in summer; among pedigree dogs, in hounds, gundogs, and
non-sporting breed dogs; and in puppies less than 6 months of
age.47 Vomiting and lethargy at admission as well as lympho-
penia and hypoalbuminemia have been associated with pro-
longation of hospitalization times by approximately 2 days.29
Positive changes in leukocyte counts (and especially lymphocyte
counts) as early as 24 hours after admission have also been asso-
ciated with survival.48 In general, puppies that survive the first
3 to 4 days of treatment make complete recoveries. Complications
of infection include bacteremia and septic shock, intestinal intus-
susception, aspiration pneumonia, and esophageal strictures.
Immunity and Vaccination
Immunity that follows natural infection by CPV-2 variants is
probably lifelong. Immunization with attenuated live viral vac-
cines also provides sterile immunity that may even be lifelong.
Both attenuated live and inactivated CPV vaccines are available.
With the possible exception of shelter environments, attenuated
live vaccines should never be administered to pregnant bitches
because they may cause disease in the developing fetus. If possible,
pregnant bitches introduced into shelter environments should be
tested for antibody to CPV before vaccination with attenuated live
vaccines, and these dogs should only be vaccinated if they test
negative. The use of inactivated vaccines is not recommended in
contaminated environments, because the window of vulnerability
and the time to onset of protection is too long. The window of
vulnerability is the period when maternal antibody interferes with
the vaccine’s ability to stimulate an effective immune response,
but does not prevent infection with virulent field virus (see Chap-
ter 12 for a discussion of this concept). Small quantities of attenu-
ated live vaccine virus are shed from the intestinal tract after
immunization, but this should not be relied on to immunize other
in-contact animals. Protection against challenge with field virus
occurs for at least 3 years and possibly for life after the initial
puppy series is administered (see Chapter 12). Exposure of dogs
to virus in the environment may also serve to booster immunity
after vaccination.
The initial immunization series should be given every 3 to 4
weeks from 6 to 8 weeks of age, until no sooner than 14 to 16
weeks (16 to 20 weeks in breeding kennels), because of persis-
tence of sufficient titers of maternal antibody in some puppies
until this age. After that, a booster should be given at 1 year
of age, and every 3 years thereafter (see Appendix ). Puppies
should be isolated in the home environment until 7 to 10 days
after the last booster. Attendance at organized puppy classes has
been advocated as a way to promote socialization in this period,
 CHAPTER 14  Canine Parvovirus Infections and Other Viral Enteritides
and currently there is no evidence that such activities increase
risk for parvoviral enteritis. Serum antibody titers of at least
1:80 as determined with hemagglutination-inhibition correlate
well with protection.
Concern has been raised that currently available CPV vac-
cines may not provide adequate protection against CPV-2c
infection, because of reports of CPV-2c enteritis in vaccinated
dogs.51 Furthermore, the serum of animals immunized against
CPV-2, CPV-2a, and CPV-2b poorly recognized CPV-2c.52
However, experimental challenge studies have demonstrated
strong protection against CPV-2c challenge when dogs are
immunized with vaccines that contain CPV-2.53,54 The extent to
which maternal antibody against non-CPV-2c strains protects
against infection with CPV-2c requires investigation.
Both inactivated and attenuated live vaccines are available
for prevention or reduction of canine enteric coronavirus infec-
tion, but because the disease is generally mild or inapparent and
primarily occurs in puppies younger than 6 weeks of age, their
use has not been generally recommended.55
Prevention
Prevention of CPV-2 enteritis requires immunization and appro-
priate quarantine, isolation, cleaning, and disinfection proce-
dures. Proper immunization is the most effective method. Puppies
that are incompletely vaccinated should not be introduced into
environments where there has been a history of parvoviral enteri-
tis and adequate environmental disinfection cannot be guaran-
teed. Although extremely resistant to disinfectants, parvoviruses
can be inactivated with a 1 in 30 dilution of household bleach;
potassium peroxymonosulfate (Trifectant, Virkon S); accelerated
hydrogen peroxide; or high-level chemical disinfectants such as
CASE EXAMPLE
Signalment: “Sam”, an 8-week old intact male Hungarian
vizsla puppy from northern California.
History: Sam was acquired 6 days ago from a breeder, and
at that time was eating and active. The next day Sam was
examined at a local veterinary clinic, and a routine fecal
examination revealed infection with Giardia and Isospora spp.
Treatment with sulfamethoxazole was initiated, but 1 day
later the puppy developed diarrhea. The next day, lethargy
and vomiting were noted. Vomiting and diarrhea occurred
every few hours. The diarrhea contained fresh blood and
the vomitus consisted of clear, frothy fluid. Inappetence and
intermittent vomiting continued for the next 2 days.
Current Medications: None
Other Medical History: Vaccination with an attenuated
live CDV, CPV-2, and canine parainfluenza vaccine was
performed when Sam was 6 weeks of age. There were no
other dogs in the household, only one adult cat.
Physical Examination:
Body weight: 4.2 kg
General: Quiet, alert, responsive. Estimated to be 7% to
8% dehydrated, T = 103.4°F (39.7°C), HR = 240 beats/min,
RR = 36 breaths/min, mucous membranes pale pink and tacky,
CRT = 2 seconds.
149
glutaraldehyde and ortho-phthalaldehyde, when contact times
of at least 10 minutes are used (see Chapter 11 for more infor-
mation on disinfection). These disinfectants will also inactivate
other enteric viruses. Steam cleaning should be used on surfaces
that cannot be otherwise disinfected, and dishwashers that attain
temperatures of at least 75°C should be used to wash dishes. For
shelters, quarantine periods of 2 weeks have been recommended
before introduction of puppies that might be shedding virus,
because shedding rarely continues for longer than 10 days.56 The
bathing of recovered puppies can help to remove virus that per-
sists on the haircoat. Rodent and insect vector control can also be
used to prevent spread of the virus in the environment. Outdoor
grassy areas and dirt are difficult or impossible to adequately
disinfect, and so only immunized animals should be allowed on
these areas. The use of bleach on these areas is not recommended
because of adverse environmental effects from runoff.
The prevalence and impact of enteric viral infections can
also be reduced through regular removal of fecal contamina-
tion, prolonged exposure of contaminated surfaces to sunlight
and drying, and elimination of stressors such as overcrowding,
transport, poor nutrition, and concurrent infections such as
intestinal parasites.
Public Health Aspects
Enteric viral pathogens of dogs, including CPV-2 variants, do
not infect humans. However, other infectious causes of gastro-
enteritis in puppies have the potential to be zoonotic, and co-
infections with these pathogens can occur, so caution should be
maintained when handling puppies with diarrhea. Precautions
used in isolation should be sufficient to prevent enteric zoonoses
(see Chapter 11).
Integument: Full, shiny haircoat. No ectoparasites were seen.
Eyes, ears, nose, and throat: Enophthalmos and a dry nasal
planum were present.
Musculoskeletal: Body condition score 2/9. The dog was
ambulatory, but emaciated and weak.
Cardiovascular: Weak but synchronous femoral pulses. No
murmurs or arrhythmias ausculted.
Respiratory: No clinically significant findings.
Gastrointestinal: No evidence of abdominal pain on palpation.
Fluid-filled intestinal tract. Full urinary bladder noted.
Rectal examination: Bloody diarrhea was present on the
thermometer. Rectal examination was not performed.
Lymph nodes: All lymph nodes were within normal limits.
Laboratory Findings:
CBC:
HCT 31.5% (40-55%)
MCV 62.6 fL (65-75 fL)
MCHC 35.6 g/dL (33-36 g/dL)
WBC 530 cells/µL (6000-13,000 cells/µL)
Neutrophils 16 cells/µL (3000-10,500 cells/µL)
Lymphocytes 504 cells/µL (1000-4000 cells/µL)
Monocytes 11 cells/µL (150-1,200 cells/µL)
Platelets clumped but appeared adequate.
Serum Chemistry Profile:
Sodium 143 mmol/L (145-154 mmol/L)
Potassium 3.8 mmol/L (3.6-5.3 mmol/L)
Continued
150 SECTION 1  Viral Diseases
Chloride 106 mmol/L (108-118 mmol/L)
Bicarbonate 23 mmol/L (16-26 mmol/L)
Phosphorus 4.8 mg/dL (3.0-6.2 mg/dL)
Calcium 10.3 mg/dL (9.7-11.5 mg/dl)
BUN 6 mg/dL (5-21 mg/dL)
Creatinine <0.2 mg/dL (0.3-1.2 mg/dL), glucose 133 mg/dL
(64-123 mg/dL)
Total protein 4.3 g/dL (5.4-7.6 g/dL)
Albumin 2.4 g/dL (3.0-4.4 g/dL)
Globulin 1.9 g/dL (1.8-3.9 g/dL)
ALT 22 U/L (19-67 U/L), AST 15 U/L (19-42 U/L)
ALP 179 U/L (21-170 U/L)
Creatine kinase 274 U/L (51-399 U/L)
Gamma GT <3 U/L (0-6 U/L)
Cholesterol 278 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL)
Magnesium 2.0 mg/dL (1.5-2.6 mg/dL).
Imaging Findings:
Abdominal radiographs: The stomach wall appeared
thickened. Numerous round gas opacities were present
in a loop of intestine in the right cranial abdomen, which
were believed to represent gas in the ascending colon
or duodenum. Multiple additional gas opacities were
present in a loop of intestine in the mid right abdomen at
the level of the fourth lumbar vertebra. There was reduced
serosal detail compatible with the age of the animal.
Changes were considered consistent with the presence of
gastroenterocolitis.
Microbiologic Testing:
Fecal Parvovirus Antigen ELISA: Negative
Fecal Enteric Real-time PCR Panel: PCR positive for CPV in
fecal specimen. PCR negative for Clostridium difficile toxin
A and toxin B genes, Cryptosporidium spp., Salmonella spp.,
and Giardia spp. DNA.
Fecal Zinc Sulfate Centrifugal Flotation: Negative for
parasite ova.
SUGGESTED READINGS
Carmichael LE. An annotated historical account of canine parvovirus.
J Vet Med Ser B. 2005;52(7-8):303-311.
Iris K, Leontides LS, Mylonakis ME, et al. Factors affecting the occur-
rence, duration of hospitalization and final outcome in canine parvo-
virus infection. Res Vet Sci. 2010;89:174-178.
REFERENCES
1. Carmichael LE. An annotated historical account of canine parvovi-
rus. J Vet Med Ser B. 2005;52(7-8):303-311.
2. Hoelzer K, Parrish CR. The emergence of parvoviruses of carni-
vores. Vet Res. 2010;41:39.
3. Clegg SR, Coyne KP, Parker J, et al. Molecular epidemiology and
phylogeny reveal complex spatial dynamics in areas where canine
parvovirus is endemic. J Virol. 2011;85:7892-7899.
4. Ikeda Y, Nakamura K, Miyazawa T, et  al. Feline host range of
canine parvovirus: recent emergence of new antigenic types in cats.
Emerg Infect Dis. 2002;8:341-346.
5. Battilani M, Balboni A, Ustulin M, et al. Genetic complexity and
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Diagnosis: Enteritis and leukopenia secondary to canine
parvoviral infection.
Treatment: Sam was admitted to isolation and treated
aggressively with intravenous lactated Ringer’s solution that
was supplemented with 20 mEq KCl/L, famotidine (0.5 mg/kg
IV q12h), ondansetron (0.5 mg/kg IV q12h), ampicillin (22 mg/
kg IV q6h), and enrofloxacin (5 mg/kg IV q24h). Because of
persistent vomiting, maropitant citrate (1 mg/kg IV q24h) was
added as a second antiemetic. Blood glucose concentration
was monitored three times daily, but supplementation with
dextrose was not required. Bloody diarrhea that contained
sloughed intestinal mucosa occurred every few hours for
the first 24 hours. The neutrophil count was 9 cells/µL the
following day, but by day 4 of hospitalization was 261 cells/µL,
with 241 moderately toxic bands/µL and a lymphocyte count
of 1226 cells/µL. There was gradual clinical improvement
during this time, and Sam began to ingest small quantities
of cottage cheese and rice on day 4. He was discharged from
the hospital on day 5.
Comments: Despite the negative fecal antigen ELISA assay,
CPV enteritis was suspected in this dog based on the history,
clinical signs, and severe leukopenia. The finding of leukopenia
and diarrhea is not diagnostic for parvoviral enteritis, because
it can also be caused by other severe enteritides such as
salmonellosis. The positive fecal real-time PCR assay for CPV
supported the diagnosis, but because positive fecal PCR
results can occur in healthy dogs with some assays, and also
after vaccination, the fecal PCR result alone did not confirm the
diagnosis of CPV enteritis. The diagnosis was therefore made
on the basis of the combination of findings. The neutropenia
in this dog was profound. Infection likely occurred before the
owner acquired the puppy because the diarrhea began only 2
days after the puppy was purchased.
6. Pratelli A, Buonavoglia D, Tempesta M, et  al. Fatal canine par-
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7. Ohshima T, Kawakami K, Abe T, et al. A minute virus of canines
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8. Eminaga S, Palus V, Cherubini GB. Minute virus as a possible cause
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Assoc. 1996:542-546.
10. Glickman LT, Domanski LM, Patronek GJ, et  al. Breed-related
risk factors for canine parvovirus enteritis. J Am Vet Med Assoc.
1985;187:589-594.
11. Kalli I, Leontides LS, Mylonakis ME, et  al. Factors affecting the
occurrence, duration of hospitalization and final outcome in canine
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12. Brady S, Norris JM, Kelman M, et al. Canine parvovirus in Aus-
tralia: the role of socio-economic factors in disease clusters. Vet J.
2012;193:522-528.
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13. Li L, Pesavento PA, Shan T, et al. Viruses in diarrheic dogs include
novel kobuviruses and sapoviruses. J Gen Virol. 2011;92:2534-2541.
14. Pesavento PA, Li L, Leutenegger C, et-al. Canine circovirus is asso-
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15. Ntafis V, Xylouri E, Radogna A, et al. Outbreak of canine norovi-
rus infection in young dogs. J Clin Microbiol. 2010;48:2605-2608.
16. Mesquita JR, Barclay L, Nascimento MS, et al. Novel norovirus in
dogs with diarrhea. Emerg Infect Dis. 2010;16:980-982.
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lar characterization of a canine norovirus. Emerg Infect Dis.
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puppy mortality without evidence of concurrent canine parvovirus
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rus enteritis 2: pathogenesis. Vet Rec. 1984;115:453-460.
20. Otto CM, Rieser TM, Brooks MB, et  al. Evidence of hypercoag-
ulability in dogs with parvoviral enteritis. J Am Vet Med Assoc.
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22. Lenghaus C, Studdert MJ, Finnie JW. Acute and chronic canine
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25. Lenghaus C, Studdert MJ. Generalized parvovirus disease in neona-
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28. Agungpriyono DR, Uchida K, Tabaru H, et  al. Subacute mas-
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1999;36:77-80.
29. Iris K, Leontides LS, Mylonakis ME, et  al. Factors affecting the
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30. Woldemeskel M, Liggett A, Ilha M, et  al. Canine parvovirus-2b-
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31. Favrot C, Olivry T, Dunston SM, et  al. Parvovirus infection of
keratinocytes as a cause of canine erythema multiforme. Vet Pathol.
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32. Decaro N, Desario C, Beall MJ, et al. Detection of canine parvovi-
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33. Schmitz S, Coenen C, Konig M, et al. Comparison of three rapid
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36. Marulappa SY, Kapil S. Simple tests for rapid detection of canine
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38. Mochizuki M, Ohshima T, Une Y, et al. Recombination between
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39. Nho WG, Sur JH, Doster AR, et al. Detection of canine parvovirus
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CHAPTER 15
Canine Distemper Virus Infection
Jane E. Sykes
Overview of Canine Distemper
First Described: 1905, Henri Carré, France1
Cause: Canine distemper virus (family Paramyxoviridae, sub-
family Paramyxovirinae, genus Morbillivirus)
Affected Hosts: Dogs and other Canidae such as coyotes,
foxes, and wolves; Procyonidae (raccoons, pandas); Mus-
telidae (ferrets, mink, skunks, otters). Large wild Felidae
can also be affected.
Geographic Distribution: Worldwide
Mode of Transmission: Oronasal contact with virus in secre-
tions or excretions, droplet nuclei, and large particle aero-
sol transmission
Major Clinical Signs: Fever, lethargy, inappetence, vomit-
ing, diarrhea, dehydration, tachypnea, cough, conjunc-
tivitis, neurologic signs, footpad, and nasal planum
hyperkeratosis
Differential Diagnoses: Canine parvovirus infection, other
infectious causes of gastroenteritis, other causes of
canine infectious respiratory disease, rabies, parasitic gas-
trointestinal and respiratory disease, toxins such as lead
and ethylene glycol, gastrointestinal foreign body, dietary
indiscretion, protozoal meningoencephalitis, systemic
fungal infections such as cryptococcosis, portosystemic
shunting with hepatic encephalopathy
Human Health Significance: CDV has been used as a model to
study measles virus infection. There is some evidence that
it may be involved in the pathogenesis of Paget’s disease,
a chronic, focal skeletal disorder of the elderly.
Etiology and Epidemiology
Canine distemper is an important disease of domestic dogs
and wild animals worldwide. It is caused by canine distem-
per virus (CDV), an enveloped, pleomorphic RNA virus that
belongs to the genus Morbillivirus (family Paramyxoviridae).
CDV is closely related to human measles virus and rinderpest
virus and has been used as a model to study the pathogenesis of
measles virus infections. The outer envelope of CDV contains
hemagglutinin (H) and fusion (F) proteins, which are impor-
tant in cellular attachment and entry (Figure 15-1). Infection of
dogs can lead to a severe, multisystemic disease that primarily
affects the gastrointestinal, respiratory, and neurologic systems
(Figure 15-2).
152
Ferrets also are highly susceptible to distemper, but domestic
cats are not affected. In addition, distemper occurs in a variety
of wildlife species that belong to the families Canidae, Musteli-
dae, Procyonidae, and Felidae (see Overview). Virus shed by
wildlife species such as raccoons can infect domestic dogs, but
virus that is shed by dogs may also be a significant threat to
wildlife populations, with catastrophic outbreaks of disease and
high mortality rates. In domestic dogs with signs of infectious
upper respiratory disease (“kennel cough”), distemper is an
important differential diagnosis, and distemper can also mimic
canine parvovirus (CPV) enteritis. In fact, dual infections with
CDV and CPV are probably underrecognized.
Worldwide, at least eight different geographic lineages
(also referred to as genotypes or clades) of CDV exist based
on sequence analysis of the H gene: Asia-1, Asia-2, America-1,
America-2, Europe-1/South America-1, Europe-2 (European
wildlife), Europe-3 (Arctic-like), and South Africa. In addition,
strains recognized in Argentina, Africa, Asia, and Mexico may
represent separate geographic lineages.1,2 In the United States,
vaccine strains belong to the America-1 lineage, whereas most
field strains belong to the America-2 lineage. Strains that belong
to the Europe-2 and Europe-3 lineages have also been detected
in North American dogs.3
Although vaccination has reduced the incidence of the dis-
ease, distemper still remains important where large numbers of
Fusion protein
Hemagglution
SH protein
Matrix protein
Nucleoprotein
Polymerase
FIGURE 15-1  Structure of CDV. The outer envelope contains hemagglutinin (H) and
fusion (F) proteins, which are connected to the nucleocapsid by a matrix (M) protein, which
is the most abundant protein in the virion.
 CHAPTER 15  Canine Distemper Virus Infection 153

FIGURE 15-2  Anatomic sites targeted by CDV infection.

young dogs with inadequate immunity are housed together, such

as in kennels, large breeding facilities, and shelter environments.
Puppies that have lost their maternal antibody are predisposed
in regions where vaccination is performed, but occasionally dis-
ease occurs in older, vaccinated dogs.4-6 Distemper also occurs
in regions where vaccination of dogs is not performed or is
poorly timed. As an enveloped virus, CDV is susceptible in the
environment (surviving less than 1 day at room temperature)
and is readily inactivated by heat, drying, and disinfectants;
thus, contact between infected dogs is important to maintain
transmission of the virus.

Clinical Features
Signs and Their Pathogenesis
CDV is highly contagious and is spread through droplet nuclei and
large-particle aerosol transmission. Dogs are generally exposed
to CDV through contact with infected oronasal secretions, which
may be shed by subclinically or clinically affected dogs. The virus
initially infects monocytes within lymphoid tissue in the upper
respiratory tract and tonsils and is subsequently disseminated via
the lymphatics and blood to the entire reticuloendothelial sys-
tem. The viral hemagglutinin binds to a molecule on the sur-
face of host cells known as the signaling lymphocytic activation
molecule (SLAM, also known in humans as CD150). SLAM is a
membrane glycoprotein of the immunoglobulin superfamily. It is
important for cell entry by CDV as well as other morbilliviruses
and is a key molecule in the pathogenesis of disease.7 SLAM is
expressed by immature thymocytes, activated lymphocytes, mac-
rophages, and dendritic cells. Direct viral destruction of a sig-
nificant proportion of the lymphocyte population, and especially
CD4+ T cells, occurs within the blood, tonsils, thymus, spleen,
lymph nodes, bone marrow, mucosa-associated lymphoid tissue,
and hepatic Kupffer cells (Figure 15-3).8-10 Massive destruction
of lymphocytes results in an initial lymphopenia and transient
fever, which occurs a few days after infection. Subsequently,
there is a second stage of cell-associated viremia and fever
FIGURE 15-3  Lymph node histopathology of a 5-month-old German shepherd
mix with distemper. The dog had been adopted from a shelter and subsequently devel-
oped neurologic signs, diarrhea, and pneumonia. There is marked lymphoid necrosis.
H&E stain.
(8 to 9 days after infection), after which CDV infects cells of the
respiratory, gastrointestinal tract, central nervous system (CNS),
urinary tract, and skin, as well as red and white blood cells,
including additional lymphoid cells. In this stage of infection,
CDV infects a variety of cell lineages, including epithelial, mes-
enchymal, neuroendocrine, and hematopoietic cells, and forms
intracytoplasmic but also intranuclear inclusions (Figure 15-4).
Infection of epithelial cells by the virus is inefficient and occurs
relatively late in the course of infection, through a SLAM-inde-
pendent mechanism that appears to involve the nectin 4 receptor,
as is true for measles virus.11 CDV is shed in all secretions and
excretions from as early as day 5 after infection, before the onset
of clinical signs. Shedding of virus can continue for as long as
3 to 4 months, but usually resolves after 1 to 2 weeks.
154 SECTION 1  Viral Diseases

A B
FIGURE 15-4  A, Eosinophilic intracytoplasmic and intranuclear viral inclusions (arrows) in the lung of the dog from Figure 15-3. Hematoxylin and eosin stain, 1000× oil magnification.
B, Intracytoplasmic inclusions (arrows) in a circulating leukocyte of a 4-month-old Border collie mix with distemper.

The clinical signs of distemper in dogs vary dramatically

and are highly dependent on virus strain and the age and
immune status of the host, as well as concurrent infections
with other viruses and bacteria. Many dogs experience sub-
clinical infection, whereas others experience rapidly progres-
sive infection followed by death. The incubation period ranges
from 3 to 6 days. Respiratory or gastrointestinal tract signs
may be indistinguishable from those caused by other respira-
tory or enteric viruses and bacteria, or signs may be so mild
that they go unnoticed by the owner. Dogs with respiratory
involvement may exhibit fever, bilateral serous and nasal ocu-
lar discharges, conjunctivitis, and a nonproductive cough.
Secondary bacterial infection, which occurs unhindered as a
result of virus-induced immunosuppression, can lead to the
development of mucopurulent nasal and ocular discharges
and bacterial bronchopneumonia, with tachypnea, productive
cough, lethargy, and decreased appetite. Viral destruction of
the gastrointestinal tract epithelium can result in inappetence,
vomiting, diarrhea, electrolyte abnormalities, and dehydra-
tion. Dogs that mount an intermediate or delayed immune
response may recover from acute illness but fail to elimi-
nate the virus completely, which leads to a spectrum of more
chronic disease manifestations, which often involve the uvea,
lymphoid organs, footpads, and especially the CNS. Opportu-
nistic infections can also develop in these dogs.
Up to 30% of infected dogs develop CNS signs. These usu-
ally occur 1 to 6 weeks after the onset of acute illness but can
occur even when initial infection is subclinical. Neurologic
signs are more likely to develop following infection with cer-
tain CDV strains, such as the Snyder Hill strain.12 The types
of brain cells that are infected differ with the strain of CDV
involved,13 but neuronal necrosis and atrophy can occur.14
Neurologic signs are often progressive and generally do not
resolve, so dogs that recover often have residual neurologic
deficits. “Old dog encephalitis” is a poorly characterized,
progressive immune-mediated demyelinating leukoencepha-
lomyelitis induced by CDV that occurs weeks to years after
recovery from the acute infection and is difficult to diagnose.
The term “old dog encephalitis” is a misnomer, because
affected dogs are not necessarily old. Both CDV and measles
virus have been incriminated as possible agents of multiple
FIGURE 15-5  Enamel hypoplasia induced by CDV infection.
sclerosis because of the resemblance of demyelinating CDV
encephalitis to the pathology of this disease.15 Virus has been
detected within the brains of these dogs with immunohisto-
chemistry and reverse-transcriptase PCR assays.16 Persistent
infection of astrocytes in the CNS by some CDV strains has
been postulated as a mechanism for this chronic manifesta-
tion of infection, with transmission from cell to cell through
induction of cell-cell fusion and resultant evasion of the host
immune response.17
Ocular signs of persistent CDV infection include uveitis, cho-
rioretinitis, keratoconjunctivitis sicca (KCS), keratitis, and optic
neuritis, which may be associated with blindness. KCS is thought
to result from damage to the lacrimal gland by CDV.18 It may
be transient or permanent, and can lead to keratitis and corneal
ulceration. The virus itself can also infect the corneal epithelial
and stromal cells. Enamel and dentin hypoplasia, manifested as
irregularities of the teeth, is an incidental finding in recovered
dogs due to infection of the stratum intermedium of developing
teeth (Figure 15-5). Retention and partial eruption of teeth has
also been reported as a sequela of CDV infection.19,20 Infection
of the skin can lead to a cutaneous measles-like rash, although
this is uncommonly recognized. Persistent infection of the foot-
pad and nasal planum epithelium leads to hyperkeratosis in
these regions (Figure 15-6).
 CHAPTER 15  Canine Distemper Virus Infection 155

A B

C
FIGURE 15-6  Nasal planum (A) and footpad (B) hyperkeratosis induced by CDV infection in a 4-month-old Border collie mix with conjunctivitis, mucopurulent nasal and ocular
discharge, and myoclonus. Blepharospasm is also present. C, Histopathology of the footpad of a 1-year old German shepherd mix with distemper. Marked hyperkeratosis is evident. H&E
stain, 40× magnification.

Immunosuppression results from lymphopenia, necrosis
of hematopoietic cells in the bone marrow, and dendritic cell
malfunction. Lymphocyte apoptosis also occurs independent
of viral infection of lymphocytes.21 The viral V protein allows
CDV to replicate rapidly in T cells and is critical in CDV-
mediated immunosuppression. This protein almost completely
antagonizes IFN-α, TNF-α, Il-6, IFN-γ, and Il-2 in the acute
phase of infection.10 Finally, the nucleocapsid (N) protein of
morbilliviruses binds to the CD32 (Fc-γ) receptor on B cells,
which results in impaired differentiation of B cells into plasma
cells.22 When the virus binds this receptor on dendritic cells,
impaired antigen presentation by dendritic cells results, which
disrupts T-cell function. The most common secondary infections
in distemper are secondary bacterial infections that contribute
to bronchopneumonia. Bordetella bronchiseptica is a common
copathogen. Dogs may be diagnosed with bordetellosis in the
early stages of distemper, and the underlying CDV infection
may be overlooked. Other opportunistic infections that have
been identified in dogs with distemper include toxoplasmosis,23
salmonellosis,24 nocardiosis,25,26 and generalized demodecosis.
Infection with Pneumocystis carinii was associated with CDV
infection in a mink,27 and concurrent neosporosis and canine
distemper was reported in a raccoon.28
Uncommonly, CDV infection has been associated with
metaphyseal bone lesions in young dogs as a result of infec-
tion of osteoclasts, osteoblasts, and osteocytes.29 Infected cells
undergo degeneration and necrosis, and metaphyseal osteoscle-
rosis occurs (Figure 15-7). This may lead to pain and lameness.
CDV RNA has also been detected in bone cells of dogs with
hypertrophic osteodystrophy, and CDV may play a role in this
disease.30
Finally, transplacental infections with CDV may be associ-
ated with infertility, stillbirth, or abortion and with neurologic
signs in puppies that are less than 4 to 6 weeks of age.
Physical Examination Findings
Physical examination findings in dogs with distemper depend
on the severity and chronicity of the disease, but include fever
(occasionally up to 106°F or 41.1°C), conjunctivitis, serous to
mucopurulent ocular and nasal discharges, blepharospasm and
photophobia, tonsillar enlargement and hyperemia, tachypnea,
and increased lung sounds (see Figure 15-6). Cough may occur
during the examination. Dehydration may also be present, espe-
cially in dogs with gastrointestinal and severe neurologic signs.
Rarely, a measles-like cutaneous rash or pustular dermatitis is
evident.
Dogs with chronic disease can be thin or emaciated and
show hyperkeratosis of the nasal planum and footpads. Puppies
with hyperkeratosis, also known as “hardpad,” have thickened,
crusty footpads that resemble those of adult dogs (see Figure
15-6). Myoclonus (involuntary twitching of isolated muscle
groups) is common and, when mild, is most readily detected
156 SECTION 1  Viral Diseases
FIGURE 15-7  Severe, neutrophilic osteomyelitis with submetaphyseal osteone-
crosis and extensive bony resorption in a 4-month-old intact male Labrador retriever
mix with distemper. The dog was seen for lethargy, mild diarrhea, fever, and reluc-
tance to stand; joint pain was detected on physical examination. Direct IFA on urine
sediment was positive for CDV, but a conjunctival scrape tested negative with direct
IFA. Necropsy also revealed pneumonia with intracytoplasmic and intranuclear viral
inclusions. H&E stain.
when affected puppies are at rest. Other neurologic signs
include obtundation, seizures, tremors, opisthotonos, tetrapa-
resis, paraparesis, delayed placing reactions, ataxia, and, less
commonly, behavioral abnormalities, compulsive pacing, and
vestibular signs such as a head tilt, nystagmus, strabismus, and
circling.4 Focal seizures may be localized to the head and jaw,
with accompanying foamy hypersalivation (“chewing gum”
seizures). Vocalization and apparent blindness can also occur.
In addition to conjunctivitis and ocular discharge, an oph-
thalmologic examination may reveal corneal edema, corneal
ulceration, aqueous flare, chorioretinitis, and uveitis, or optic
neuritis. Markedly decreased Schirmer tear test results and posi-
tive fluorescein tests may be present.18 Optic neuritis appears as
a large, pale, fluffy, optic disc with no physiologic depression.
Rarely, lameness may be observed.
Dogs that have recovered from CDV infection may have
evidence of dental abnormalities and enamel hypoplasia (see
Figure 15-5); healed chorioretinitis lesions, which appear as
hyperreflective circular lesions (“gold medallion lesions”); kera-
toconjunctivitis sicca; or residual neurologic signs, especially
myoclonus.
Diagnosis
Establishment of a diagnosis of distemper is most easily accom-
plished in dogs that have the full spectrum of clinical abnormali-
ties, and especially when myoclonus is present, because there
are few other diseases that cause this array of clinical signs.
In dogs with isolated respiratory, gastrointestinal, or neuro-
logic signs, antemortem diagnosis is more challenging because
these signs are less specific, laboratory diagnostic assays can be
insensitive, and the presence of attenuated live vaccine virus
can lead to false-positive results. A combination of laboratory
tests may be required to establish the diagnosis, and the finding
of a negative result for any assay does not rule out distemper.
Sensitivity for detection of CDV using assays that detect virus
may also be increased by testing of multiple specimens from
different anatomic locations, such as various combinations of
blood, urine, conjunctival smears, and CSF. In dogs with respi-
ratory signs, the presence of conjunctivitis, blepharospasm, and
ocular discharge should raise suspicion for distemper. Distem-
per should always be considered as a diagnosis in young dogs
with neurologic signs, whether or not respiratory or gastroin-
testinal signs are present. Finally, clinicians should maintain a
high suspicion for the presence of concurrent infections such
as parvoviral enteritis, concurrent respiratory viral infection, or
bordetellosis.
Laboratory Abnormalities
Complete Blood Count
The CBC in dogs with acute distemper most commonly reveals
mild anemia and lymphopenia (Table 15-1). However, the lym-
phocyte count may also be normal, especially with more chronic
distemper. Neutropenia, monocytopenia, and thrombocytope-
nia can occur, and occasionally these are severe. Neutrophilia
with a left shift and toxic neutrophils may be present. Rarely,
examination of Romanowsky-stained blood smears using light
microscopy reveals CDV inclusions in the cytoplasm of circulat-
ing erythrocytes and leukocytes (see Figure 15-4, B).
Serum Biochemical Tests
Abnormalities on the serum biochemistry panel in dogs with
distemper are nonspecific and include electrolyte changes that
occur with vomiting and diarrhea such as hyponatremia, hypo-
kalemia, and hypochloridemia (Table 15-2). Hypoalbuminemia
and associated hypocalcemia are also common. Mild increases
in liver enzyme activities occur in some dogs, which may reflect
hypoxia or secondary infections that occur as a result of trans-
location of intestinal bacteria.
Urinalysis
No specific urinalysis findings are reported in dogs with
distemper.
CSF Analysis
CSF analysis may show no clinically significant findings in dogs
with distemper, especially when chronic demyelinating enceph-
alitis is present. In some dogs with CNS involvement, a mild
to moderate increase in CSF protein concentration is present
(usually <100 mg/dL), and there is lymphocytic or monocytic
pleocytosis, sometimes with reactivity. Nucleated cell counts are
usually less than 50 cells/µL and rarely less than 100 cells/µL.4
Diagnostic Imaging
Plain Radiography
When present, abnormalities on thoracic radiography in dogs
with distemper include pulmonary interstitial to alveolar infil-
trates and consolidation compatible with bronchopneumonia,
especially in the cranial and ventral lung lobes.
Magnetic Resonance Imaging
Dogs with distemper encephalomyelitis can have normal mag-
netic resonance imaging findings. Hyperintense lesions and loss
of contrast between gray and white matter in the cerebellum and/
or the brainstem have been described in T2-weighted images,
which correspond to demyelination on histopathology.31 Multi-
focal T2 and fluid-attenuated inversion recovery (FLAIR) hyper-
intensities can occur in other locations in the brain, and there
may be mild meningeal contrast enhancement.
 CHAPTER 15  Canine Distemper Virus Infection 157

TABLE 15-1
Complete Blood Count Findings at Admission in 50 Dogs Diagnosed with Distemper at the UC Davis VMTH
Percent below Percent within Percent above Range for
Reference the Reference the Reference the Reference Dogs with
Test Range Range Range Range Distemper
Hematocrit (%) 40-55 80 20 0 17-53
MCV (fL) 65-75 26 72 2 58-80
MCHC (g/dL) 33-36 10 74 16 28-37
Neutrophils (cells/µL) 3000-10,500 12 32 56 16-27,765
Band neutrophils 0-rare 0 72 28 0-4442
(cells/µL)
Monocytes (cells/µL) 150-1200 4 62 34 24-5202
Lymphocytes (cells/µL) 1000-4000 54 42 4 110-5575
Eosinophils (cells/µL) 0-1500 0 100 0 0-1114
Platelets (cells/µL) 150,000-400,000 14 66 20 17,000-594,000

Note: Adult reference ranges were used.

TABLE 15-2
Findings on Serum Biochemistry Analysis in 27 Dogs with Distemper at the UC Davis VMTH
Percent below Percent within Percent above Range for
Reference the Reference the Reference the Reference Dogs with Number of
Test Range Range Range Range Distemper Dogs Tested
Sodium (mmol/L) 145-154 56 40 5 106-158 26
Potassium (mmol/L) 3.6-5.3 12 79 9 2.8-5.9 26
Chloride (mmol/L) 108-118 40 56 5 65-130 26
Bicarbonate (mmol/L) 16-26 0 86 14 16-34 26
Calcium (mg/dL) 9.7-11.5 25 68 7 5.1-12.7 27
Phosphorus (mg/dL) 3.0-6.2 7 50 43 2.6-10.0 27
Creatinine (mg/dL) 0.3-1.2 11 89 0 0-1.2 27
BUN (mg/dL) 5-21 7 91 2 0.6-25 27
Albumin (g/dL) 3.0-4.4 68 32 0 0.9-3.7 27
Globulin (g/dL) 1.8-3.9 7 82 11 1.5-5.3 27
Cholesterol (mg/dL) 135-361 9 86 5 88-385 26
Total bilirubin (mg/dL) 0-0.2 0 80 20 0-0.8 26
ALT (U/L) 19-67 7 75 18 8-389 26
ALP (U/L) 21-170 0 77 23 28-322 26

Note: Adult reference ranges were used by the laboratory.

Microbiologic Tests distinguished from vaccine virus in dogs that have been recently
Microbiological tests for CDV infection are shown in Table 15-3. vaccinated with attenuated live vaccines.

Virus Isolation Cytologic Demonstration of CDV Inclusions

CDV can be readily isolated in SLAM transfected Vero cell Intracytoplasmic viral inclusions can occasionally be seen in
lines, which show cytopathic effects within 24 hours of inocula- circulating blood cells of affected dogs, especially early in
tion. Isolation is generally used as a research tool rather than infection (see Figure 15-4, B). Inclusions may also be visible
for commercial diagnostic purposes. Wild-type virus must be in the cytoplasm of conjunctival epithelial cells. Epithelial
158 SECTION 1  Viral Diseases
cells can be scraped from the conjunctiva using a curette
after application of a topical ophthalmic local anesthetic
preparation. The cells are smeared onto a glass slide, stained
with a Romanowsky stain and examined with light micros-
copy. Unfortunately, this has low sensitivity for diagnosis of
distemper.
Immunostaining for CDV Antigen
Direct immunofluorescent antibody (IFA) techniques are often
used in practice to detect CDV antigen in a variety of cell types,
including conjunctival epithelial cells, leukocytes in a buffy coat
smear, urine sediment, or tissues obtained at necropsy. Immu-
noperoxidase antibody can also be used. Immunostaining is
more sensitive than identification of CDV inclusions with rou-
tine stains, but false negatives still occur when low quantities of
virus are present in the specimen.
Transient false positive results might occur after vaccination
with attenuated live vaccines, so the results must be interpreted
with caution in recently vaccinated dogs. False positives also
have the potential to occur if nonspecific fluorescence is inter-
preted as a positive result by inexperienced laboratory techni-
cians. Further research is required to understand the prevalence
of, and influence of vaccination on false positive direct IFA
results for diagnosis of distemper.
Antigen Detection ELISA Assays
ELISA assays that detect CDV antigen in serum (Table 15-3) are
available commercially as in-practice kits in some countries. The
sensitivity and specificity of these assays has not been well stud-
ied, and there are no published reports in the scientific literature
of the performance of commercially available assays, only those
in the research and development phase. One assay had a low
sensitivity in dogs that were naturally infected with distemper,
with positive results in only 27% of 26 dogs at initial exami-
nation and 12% when the dogs were examined 2 to 3 weeks
later.32 False-positive results occurred for up to 4 weeks after
vaccination in 20% of 40 dogs immunized with attenuated live
viral vaccines. Another lateral flow (immunochromatography)
assay had sensitivity equivalent to that of an RT-PCR assay
when applied to conjunctival swab specimens, but sensitivity
was 90% and 86% when the assay was applied to blood lym-
phocytes and nasal swab specimens from dogs with distemper.33
Molecular Diagnosis Using the Polymerase Chain Reaction
Reverse transcriptase-PCR (RT-PCR) assays are offered by a
number of commercial veterinary diagnostic laboratories for
diagnosis of distemper, sometimes as part of a panel that includes
assays for other canine respiratory pathogens (see Chapter 17).
Most assays detect a portion of the N protein gene. RT-PCR
can be performed on whole blood, buffy coat, skin biopsies,
conjunctival scrapings, urine, CSF, transtracheal washes, nasal
and oropharyngeal swabs, and a variety of other tissues collected
at necropsy (e.g., lung, small intestine, stomach, kidney, brain,
bladder, lymphoid tissues). In one study of a limited number of
experimentally infected dogs, conjunctival swabs were superior
to whole blood, nasal flushes, and urine for PCR diagnosis of
CDV infection,34 whereas another study of naturally infected
dogs showed highest viral loads in blood, followed by conjunc-
tival swabs, and then urine.35 RT-PCR is more sensitive than
direct IFA, but false positives may be more likely to occur after
vaccination with attenuated live virus vaccines, because RT-PCR
assays that are currently available for routine diagnostic purposes
do not distinguish between vaccine and wild-type strains. Assays
have been developed that differentiate between field and vaccine
strains of CDV.36 False-positive test results should not occur
after use of recombinant CDV vaccines. More research is needed
to clarify the influence of vaccination on the results of RT-PCR
for distemper. Quantitation of RT-PCR results may ultimately
be helpful in differentiating vaccination with attenuated virus
­vaccines (low viral load) and natural infection (high viral load).
False-negative RT-PCR results can occur if the virus is pres-
ent in low levels at the site from which specimens are collected.
Because RNA is labile, degradation of viral RNA during speci-
men collection and transport to the laboratory can also lead to
false-negative PCR results. In outbreak situations, collection of
specimens from multiple dogs (at least 5 to 10) may increase the
chance of positive test results.37 The diagnosis is strengthened
when positive PCR assay results are combined with positive
results of other assays such as direct IFA.
Serology
Antibodies to CDV can be measured in the laboratory using
serum neutralization (SN) assays (see Chapter 2). ELISA assays
that detect IgG and IgM are also available for routine diagnosis
of distemper in dogs, which have improved sensitivity and speci-
ficity over SN.38 In one study, IgM antibodies appeared 1 week
before SN titers.39 Use of serology for diagnosis of distemper is
complicated by the confounding effect of vaccination. This is
especially true in shelter environments where all dogs are vacci-
nated on intake. Nevertheless, a fourfold rise in titer over a 2- to
4-week period supports recent infection. Acute and convalescent
sera should be submitted to the same laboratory, because inter-
laboratory variation in assay results occurs. Ideally, an aliquot
of the acute serum specimen should be retested at the same time
the convalescent titer is determined. Antibodies within the CSF
can be quantified and compared to those in serum to determine
whether local production of antibody in the CSF is likely. Dogs
with delayed-onset CDV encephalomyelitis have high levels of
antibody in the CSF when compared with serum.40
The results of serologic tests can be used to assess the need
for vaccination, and also to determine which dogs are protected
and therefore of low risk for development of disease and to some
extent, virus shedding in outbreak situations. Serum neutraliza-
tion titers of at least 1:16 to 1:20 correlate with protection after
vaccination. Titers of 1:100 or more correlate with protection in
puppies that have received maternal antibodies.41 Because of the
presence of cell-mediated immunity, negative serologic results
do not imply lack of protection. Point-of-care ELISA assays are
also available for semiquantitative measurement of antibodies
to CDV. One assay (Synbiotics TiterCHEK CDV/CPV) had a
sensitivity of 76% and a specificity of 92% compared with SN
when performed as a point-of-care assay.42 The low sensitivity
would tend to overestimate the need for vaccination, which is
preferable to an assay with low specificity, which might result
in unnecessary exposure of a susceptible dog to infected dogs.
Pathologic Findings
Gross Pathologic Findings
In addition to physical examination abnormalities described ear-
lier, gross pathologic findings in canine distemper include thymic
atrophy, pulmonary congestion and consolidation, liquid intesti-
nal contents, and lymph node congestion and enlargement. Less
common findings are mild pleural, pericardial, and/or peritoneal
effusion and, uncommonly, visceral congestion and ecchymotic

TABLE 15-3
Diagnostic Assays Available for Distemper in Dogs
Assay Specimen Type
Virus isolation Conjunctival and nasal
swabs, blood, urine, trans-
tracheal wash specimens,
necropsy specimens
Direct immunostaining Smears made from con-
(such as direct fluorescent junctival scrapings, urine
antibody) sediment, respiratory lavage
specimens, impression
smears of tissue specimens
Distemper ELISA antigen See Virus isolation
assay
RT-PCR See Virus isolation
Serology (IgG and IgM Serum and/or CSF
ELISA; SN)
Histopathology Usually necropsy specimens,
but also skin biopsies (such
as footpad biopsies)
CDV, Canine distemper virus; RT, reverse transcriptase; SN, serum neutralization.
CHAPTER 15  Canine Distemper Virus Infection 159
Target Performance
CDV False negatives can occur in specimens
that contain low numbers of virus
particles. Attenuated live vaccine
virus may also be isolated. Not
widely offered for routine diagnostic
purposes.
CDV antigen False negatives can occur in specimens
that contain low numbers of virus
particles. False positives can occur if
nonspecific fluorescence is incorrectly
interpreted as a positive result. Pos-
sibility of false positives after recent
vaccination with attenuated live CDV
vaccines.
CDV antigen Inexpensive and rapid. Sensitivity and
specificity in naturally infected dogs
have not been well established. False
positives can occur for weeks after
vaccination with attenuated live
CDV vaccines.
CDV RNA Sensitivity and specificity can vary
depending on assay design. Testing
specimens from multiple anatomic
sites or combining PCR assays with
other diagnostic tests for CDV
improves sensitivity. Attenuated live
vaccine virus may be detected for days
to weeks after vaccination. Because
of the high sensitivity of some assays,
the significance of a positive result in
relation to disease may be difficult to
interpret. False-negative results can
occur when virus levels are low or as
a result of degradation of viral nucleic
acid during specimen transport.
Antibodies against Acute and convalescent sera are
CDV antigens required for diagnosis of acute dis-
temper, so diagnosis is retrospective.
False positives can occur with recent
CDV vaccination.
Eosinophilic intracy- If available, in situ hybridization may
toplasmic and intra- be most sensitive for detection of
nuclear inclusions; virus in tissues.
CDV antigen with
immunostaining or
CDV RNA with in
situ hybridization
160 SECTION 1  Viral Diseases
FIGURE 15-8  Bronchial epithelium from a dog infected with CDV. Intracytoplas-
mic inclusions are abundant (arrows). H&E stain. (Image courtesy Dr. Patricia Pesavento,
University of California, Davis Veterinary Anatomic Pathology service.)
FIGURE 15-9  Immunohistochemistry that shows CDV antigen (brown staining) in
bladder transitional epithelial cells from a 2-year-old male neutered Australian cattle dog
infected with CDV that had been recently adopted from a shelter and developed cough,
ataxia, blindness, and seizures.
hemorrhages and meningeal congestion. In some dogs, gross
necropsy abnormalities are minimal. Concurrent external and
intestinal parasitic infections may also be identified.
Histopathologic Findings
Histopathologic findings in the brain and spinal cord are vari-
able and depend on the CDV strain, the immune status of the
host, the presence of secondary infections, and disease chro-
nicity. Findings in the brain and spinal cord include neuronal
necrosis and degeneration, and demyelination with subacute to
chronic infection. Multifocal gliosis, astrocytosis, vacuolization,
and lymphoplasmacytic perivascular cuffing may be present. The
virus has a predilection for white matter of the lateral cerebellar
peduncles, the cerebellum, and the dorsolateral medulla adjacent
to the fourth ventricle, but lesions can also occur elsewhere, such
as the thalamus, midbrain, and pyriform lobes of the cerebral
cortex. Less commonly, nonsuppurative meningitis is observed.
Infection of the lungs leads to a lymphocytic and histiocytic inter-
stitial pneumonia with proliferation of the alveolar epithelium;
neutrophilic bronchopneumonia occurs as a result of secondary
bacterial infection. There is often widespread lymphoid deple-
tion and necrosis in all reticuloendothelial tissues (see Figure
15-3), although lymphoid hyperplasia may be observed in dogs
with chronic distemper. Epithelial cell necrosis may be noted in
the dental ameloblast layer, trachea, and bladder mucosa.
The diagnosis of CDV infection is supported by the identifi-
cation of eosinophilic intracytoplasmic or intranuclear inclusion
bodies, which are 1 to 5 µm in diameter and occur in a variety of
cell types, but especially in neurons, astrocytes, microglial cells,
conjunctival epithelial cells, bladder epithelium, footpad epi-
thelium, and sometimes lymphocytes (Figure 15-8). Less com-
monly, syncytia may be identified, such as within the meninges,
and lymph nodes. Confirmation of the presence of CDV in tis-
sues can be established with the use of immunostaining, in situ
hybridization, or RT-PCR (Figure 15-9).
Treatment and Prognosis
When required, the mainstay of treatment for CDV infection is
supportive care. No specific antiviral drug is available for treat-
ment of CDV infection, although ribavirin and 5-ethynyl-1-β-
d-ribofuranosylimidazole-4-carboxamide (EICAR) have shown
efficacy in  vitro43 and for treatment of measles in a rodent
model.44 The pharmacokinetics and toxicity of these drugs in
dogs require further study.
Dogs with mild respiratory or gastrointestinal distemper
may recover spontaneously without treatment. Dogs with
severe respiratory and gastrointestinal disease may require hos-
pitalization and treatment with IV fluids, antimicrobial drugs
for secondary bacterial pneumonia, oxygen supplementation,
nebulization, and coupage. Ideally, these dogs should be housed
in isolation, although in many veterinary hospitals, isolation
may not permit treatment with oxygen and adequate monitor-
ing. Under these circumstances, dogs could be housed under
strict barrier precautions where oxygen and critical care can be
provided (see Chapter 11). When possible, collection of a trans-
tracheal lavage specimen for culture and susceptibility is recom-
mended for dogs with secondary bacterial pneumonia. When
this is not possible, the use of antimicrobial drugs likely to be
effective against Bordetella bronchiseptica (such as doxycycline)
is recommended (see Chapter 38). Dogs with severe broncho-
pneumonia may require treatment with parenteral broad-spec-
trum antimicrobial drug combinations, such as ampicillin and
a fluoroquinolone. Other supportive treatments include enteral
nutrition, artificial tears for dogs with KCS, and antiemetics.
Parenteral nutrition should be used only if absolutely necessary,
because of the immunosuppression that occurs with CDV infec-
tion and possibility of nosocomial infections. Vitamin A sup-
plementation before experimental infection reduced morbidity
and mortality associated with CDV infection in ferrets,45 but its
usefulness as a treatment for canine distemper requires further
investigation. Administration of daily oral vitamin A for 2 days
to children younger than 2 years of age with measles reduces the

risk of mortality and pneumonia-specific mortality.46 Ascorbic
acid has also been advocated as a treatment for distemper in
dogs, but its efficacy remains unproven.41
Owners of dogs with respiratory and gastrointestinal illness
should be warned that CNS signs have the potential to develop
weeks to even months after apparent recovery. Unfortunately,
the development of CNS signs is unpredictable. When neuro-
logic signs occur, they rarely resolve and may be progressive. The
prognosis for dogs with severe neurologic signs is poor, but some
neurologic signs, such as mild myoclonus, may still be compatible
with normal daily activities and do not interfere with quality of
life. Euthanasia is recommended for dogs with neurologic signs
that are progressive and not compatible with a good quality of life.
A single IV dose of dexamethasone or tapering antiinflam-
matory doses of glucocorticoids has been advocated to halt or
reduce the severity of progressive CNS signs,41 although con-
trolled prospective studies are required to fully evaluate the
usefulness of this treatment in dogs with distemper. Anticon-
vulsants such as diazepam and, for chronic management, potas-
sium bromide or phenobarbital can be used to treat seizures and
prevent the spread of a seizure focus. However, they do not pre-
vent progressive CDV infection of the CNS and are sometimes
poorly effective. Mexiletine and procainamide have also been
suggested as treatments for distemper myoclonus, but their true
efficacy is unreported. To date, the author has had disappoint-
ing results using these treatments in a limited number of dogs
with persistent distemper myoclonus.
Immunity and Vaccination
Immunity to CDV requires antibodies as well as cell-mediated
immunity. Recovery from natural infection provides lifelong
immunity. Distemper can be effectively prevented through
immunization with attenuated live or recombinant vaccines.
Inactivated vaccines do not provide sufficient protection and
are not available in the United States. In contrast, proper vac-
cination with attenuated live vaccines can provide long-lasting
(at least 3 to 5 years), sterile immunity.47 Attenuated live vac-
cines should be given every 3 to 4 weeks, commencing no earlier
than 6 weeks of age. Maternal antibodies are usually absent
by 12 to 14 weeks of age, and so the last vaccine should be
given no sooner than 14 to 16 weeks of age (consider exten-
sion to 16 to 20 weeks in breeding kennels and shelters).47 Ide-
ally pups should be isolated for at least 7 to 10 days after the
last vaccine is administered (see Appendix). For dogs older than
16 weeks of age that are brought to the veterinarian for their
first vaccination, two doses of vaccine should be given 3 to
4 weeks apart, although a single dose may provide protection.
A booster is recommended at 1 year and then no earlier than
every 3 years thereafter.47 For shelter dogs, vaccination should
be performed as soon as possible before entry into the shelter.
A single vaccine given even hours before entry can provide pro-
tection from severe disease.48 Infection can still occur, but dis-
ease severity is lessened.
Although generally safe and effective when used according to
label recommendations, attenuated live CDV vaccine virus can
cause encephalitis when administered to immunocompromised
dogs and puppies less than 6 weeks of age, and severe disease
can occur in susceptible species such as ferrets and certain wild-
life species. Administration of attenuated live CDV vaccines to
dams immediately after whelping has been associated with the
development of encephalitis in the puppies, and it was suggested
CHAPTER 15  Canine Distemper Virus Infection 161
that vaccine virus shed by the dam may cause disease in the
pups.49 Postvaccinal encephalitis usually occurs 3 to 20 days
after vaccination and has been associated with certain batches
of vaccines that contain the Rockborn strain.50 Neurologic signs
such as ataxia may be reversible in some dogs. Seizures may be
progressive and irreversible. Some distemper vaccines available
on the market still contain the Rockborn strain, and Rockborn-
like viruses have been isolated from dogs with distemper, which
may represent residual virulence of vaccine virus or circulation
of vaccine-derived Rockborn-like viruses in the dog popula-
tion.51 Attenuated live CDV vaccines have been incriminated
as a cause of hypertrophic osteodystrophy, especially in young
Weimaraner dogs,41,52 but there is a paucity of published evi-
dence that supports the association (see Chapter 12).
A canarypox-vectored recombinant vaccine that expresses
the H and F glycoproteins of CDV is also available for preven-
tion of canine distemper (Merial Limited). This vaccine induces
a 3-year duration of immunity, protects dogs from severe disease
when administered hours before shelter admission, can protect
puppies from virulent CDV infection in the face of maternal
antibody, and can be used in pregnant bitches and puppies as
young as 4 weeks of age in the face of an outbreak or in heavily
contaminated environments.48,53 The recombinant vaccine has
replaced the use of canine measles vaccines, which were previ-
ously used to overcome maternal antibody in the face of dis-
temper outbreaks. Whether the recombinant vaccine overcomes
maternal antibody that is directed against the canarypox vector
vaccine virus as effectively as it overcomes maternal antibody
against the attenuated CDV vaccine virus requires further evalu-
ation. Further study of the relative efficacy of the recombinant
and attenuated live vaccines in heavily contaminated environ-
ments such as shelters is also required. The canarypox vectored
recombinant vaccine is also the vaccine of choice for ferrets,
and a specific ferret vaccine is available at the time of writing
(Purevax Ferret Distemper, Merial Limited).
Vaccine strains of CDV, such as Onderstepoort, Snyder
Hill, and Lederle, belong to the America-1 lineage, but field
strains that circulate in North America belong primarily to
the ­America-2 lineage. Concern has been raised that these vac-
cines may not adequately protect against infection with field
strains, but cross-neutralization studies suggest that antigenic
differences are not sufficient to warrant changes in the cur-
rent vaccines. The development of distemper in vaccinated
dogs may reflect failure of vaccine efficacy as a result of the
improper administration of vaccines, improperly timed vacci-
nation series, or the use of vaccines that have been stored and
handled inappropriately. The last is especially important for
CDV vaccines because attenuated live vaccine virus is so labile.
Vaccines should be stored in a refrigerator that maintains an
internal temperature of 0°C to 4°C and reconstituted with the
proper diluent immediately (within 1 hour) before administra-
tion. It has been recommended that vaccine held for longer than
1 hour after reconstitution be discarded.47 A second dose should
be given if some of the vaccine is found on the haircoat after
administration.
Prevention
Properly timed vaccination is the key to prevention of CDV
infection. Puppies with declining maternal antibody titers should
be kept away from other dogs until the vaccination series is
complete. In shelter situations, dogs with signs of respiratory or
162 SECTION 1  Viral Diseases
gastrointestinal disease should be held well away from other dogs
(at least 25 feet has been suggested37) and handled only after
apparently healthy dogs have been cared for. Puppies should
always be separated from adult dogs using equipment desig-
nated only for that area, and overcrowding should be prevented.
Quarantine for CDV is difficult because the incubation period
for development of neurologic signs may be as long as 6 weeks.
Wildlife species such as raccoons can also shed CDV and were
suspected as a source of infection in an outbreak that occurred in
a Chicago shelter, when raccoons and dogs were co-transported
in the back of a vehicle.37 Routine cleaning and disinfection of
contaminated surfaces readily eliminates virus in the environ-
ment. Proper nutrition may also help to prevent disease due to
CDV. Isolated vitamin A deficiency is a major risk factor for
severe measles and death in humans and distemper in ferrets. Ret-
inoids interfere with replication of measles virus in vitro through
a type I IFN-dependent mechanism.54 The impact of nutritional
status on disease due to CDV requires further investigation.
CASE EXAMPLE
Signalment: “Molly”, a 1.5-year-old female spayed golden
retriever from Sacramento, CA
History: Molly was evaluated for a 1-month-long history
of abnormal behavior that included stumbling, failure
to catch balls, and loss of interest in activity. She also had
a 1-year history of seizures, which were thought to be
due to idiopathic epilepsy and which were treated with
phenobarbital (4 mg/kg PO q12h) and potassium bromide
(17 mg/kg PO q12h). This was associated with a decrease in
seizure frequency. Two weeks after the onset of abnormal
behavior, Molly was taken to a local veterinary clinic. A
serum phenobarbital level at that time was 50 µg/mL
(therapeutic range, 10 to 40 µg/mL). The phenobarbital
dose was decreased (1.4 mg/kg PO q24h), but Molly began
circling to the left, head pressing, pacing, and vocalizing. She
also stopped responding to the owners when they called
her name, and two episodes of vomiting were noted. In the
week before evaluation, Molly refused to eat dry food, but
ate canned dog food. Her thirst had decreased and she was
voluntarily urinating in the house. A serum phenobarbital
level was repeated 2 days before she was evaluated and was
13 µg/mL. Molly had been immunized regularly (CDV, CPV,
CAV-2, and parainfluenza) and received monthly flea, tick,
and heartworm prophylaxis.
Current medications: Phenobarbital (1.4 mg/kg PO q24h);
potassium bromide (17 mg/kg PO q12h).
Physical Examination:
Body weight: 23.2 kg
General: Obtunded but hydrated. T = 101.7°F (38.7°C), HR = 60
beats/min, RR = 36 breaths/min, mucous membranes pink,
CRT = 1 sec.
Integument, eyes, ears, nose and throat: No clinically
significant abnormalities were identified. A full
ophthalmologic examination was also unremarkable.
Musculoskeletal: Body condition score was 5/9. Molly was
ambulatory on all four limbs.
Public Health Aspects
In general, CDV is not believed to infect human cells or cause
disease in people. However, both CDV and measles virus have
played a controversial role in Paget’s disease of bone, a com-
mon skeletal disorder characterized by focal abnormalities of
increased bone turnover, which is associated with bone pain,
deformity, and pathologic fractures.55 Paget’s disease most often
occurs in the elderly, men are more likely to be affected than
women, and the overall prevalence of the disease has decreased
in many geographic locations.56 Genetic factors clearly play a
role in the disease, but in some studies, the RNA of paramyxovi-
ruses that include CDV has been detected within lesions.57 Other
studies have failed to detect the nucleic acid of CDV in lesions,
and contamination has been suggested as a reason for positive
test results.55 However, CDV has been shown to infect and rep-
licate in human osteoclast precursors, further raising concern
about the possibility of zoonotic transmission of CDV.58
Cardiovascular, respiratory, gastrointestinal, and
urogenital systems: No clinically significant findings were
identified. All peripheral lymph nodes were normal sized.
Average systolic blood pressure (10 measurements) was 120
mm Hg.
Neurologic Examination:
Mentation: Obtunded and not responsive to verbal and most
tactile stimuli; head pressing.
Gait/Posture: Compulsive circling and leaning to the left was
present.
Cranial nerves: No clinically significant abnormalities were
identified.
Postural reactions: Placing reactions were absent in the left
thoracic and left and right pelvic limbs.
Segmental reflexes: Diminished biceps and withdrawal
reflexes were present, left thoracic limb only.
Panniculus: No abnormalities detected.
Spinal palpation: Pain was elicited in several regions along
the spine. Resisted cervical ventroflexion.
Neuroanatomic localization: Diffuse cerebral or thalamic
disease; C6 to T2 myelopathy.
Laboratory Findings:
CBC:
HCT 37.9% (40-55%)
MCV 68 fL (65-75 fL)
MCHC 35.4 g/dL (33-36 g/dL)
WBC 7510 cells/µL (6000-13,000 cells/µL)
Neutrophils 4859 cells/µL (3000-10,500 cells/µL)
Lymphocytes 2155 cells/µL (1000-4000 cells/µL)
Monocytes 338 cells/µL (150-1200 cells/µL)
427,000 platelets/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 141 mmol/L (145-154 mmol/L)
Potassium 4.4 mmol/L (4.1-5.3 mmol/L)
Chloride 109 mmol/L (105-116 mmol/L)
Bicarbonate 21 mmol/L (16-26 mmol/L)
Phosphorus 3.9 mg/dL (3.0-6.2 mg/dL)
Calcium 12.2 mg/dL (9.9-11.4 mg/dl)
BUN 6 mg/dL (8-31 mg/dL)
 CHAPTER 15  Canine Distemper Virus Infection 163

Creatinine 0.6 mg/dL (0.5-1.6 mg/dL) Lumbar Cisternal

Glucose 109 mg/dL (70-118 mg/dL)
Total protein 6.1 g/dL (5.4-7.4 g/dL) Large mononu- 8 20
Albumin 2.7 g/dL (2.9-4.2 g/dL) clear cells (%)
Globulin 3.4 g/dL (2.3-4.4 g/dL) Microscopic Mild to ­moderate Marginal
ALT 132 U/L (19-67 U/L) evaluation lymphocytic mononuclear
AST 62 U/L (21-54 U/L) reactivity (lymphocytic)
ALP 155 U/L (15-127 U/L) pleocytosis
Creatine kinase 399 U/L (46-320 U/L)
Gamma GT 10 U/L (0-6 U/L) Treatment and Outcome: Molly was hospitalized for a
Cholesterol 291 mg/dL (135-345 mg/dL) week during the diagnostic work-up and treated with
Total bilirubin 0.2 mg/dL (0-0.4 mg/dL). intravenous fluids and potassium bromide (35 mg/kg PO
Serum ionized calcium: 1.39 mmol/L q12h). She became progressively more obtunded and
Urinalysis: SGr 1.014; pH 7.0, few amorphous crystals. No other showed additional neurologic signs of circling to the right
abnormalities detected. and positional horizontal nystagmus with a fast phase
Serum bromide concentration: 1.1 mg/mL (therapeutic to the right. The narrowed disc spaces were thought to
range, 1.0 to 2.0 mg/mL) represent intervertebral disc disease, and granulomatous
Serum phenobarbital concentration: 1.4 µg/mL (therapeutic meningoencephalitis was suspected. Potassium bromide
range, 15 to 40 µg/mL) was discontinued, and treatment with phenobarbital (5 mg/
Serum bile acids: Pre-meal, 14 µmol/L (reference range, kg PO q12h) reinstituted. Dexamethasone was administered
0-12 µmol/L); post-meal, 16 µmol/L (reference range, 0-16 (0.2 mg/kg, IV q24h), but there was minimal improvement,
µmol/L) and euthanasia was elected.
Imaging Findings: Necropsy Findings:
Thoracic radiographs: No clinically significant abnormalities • Brain, cerebrum: severe, generalized, chronic neuronal loss,
were present. axonal necrosis, and myelin degeneration with abundant
Spinal radiographs: Disc space narrowing was present eosinophilic inclusion bodies and canine distemper antigen
between the sixth and seventh cervical vertebrae and to (immunohistochemistry)
a greater extent between the seventh cervical and first • Brain and spinal cord (C1 to T7), corticospinal tracts: severe,
thoracic vertebrae. diffuse axonal necrosis and myelin degeneration
Abdominal ultrasound: No significant lesions were identified. • Brain, cerebrum, and meninges: generalized, lymphoplas-
Magnetic resonance imaging: Precontrast: Multiple small macytic, perivascular cuffing
regions of hyperintensity were noted on dual echo and • Tonsil, lymph nodes (mandibular, mesenteric, thoracic):
FLAIR sequences, one of which was in the left thalamus mild, multifocal lympholysis with intranuclear and intracy-
along the third ventricle and another diffusely along the toplasmic eosinophilic inclusion bodies and canine distem-
lateral aspect of the right thalamus. There was a linear region per antigen
of hyperintensity along the right parietal and temporal Diagnosis: Chronic distemper encephalitis with demyelination
lobes. On T1-weighted postcontrast images, a small focus (“old dog encephalitis”)
of contrast enhancement was noted in the ventral aspect Comments: Distemper was not suspected in this dog
of the right thalamus in the region of hyperintensity on because of the dog’s older age and history of vaccination.
the precontrast fluid-weighted sequences. Impressions: Initial infection may have occurred before vaccination
Multifocal, relatively non–contrast-enhancing lesions in was complete, vaccination may have been improperly
the cerebrum and thalamus suggestive of cerebral edema, performed, or the dog may have been a vaccine
encephalitis, or small infarcts. nonresponder, which is believed to occur rarely. The
Electroencephalography: No abnormalities were detected. previous diagnosis of epilepsy further complicated the
CSF Analysis: identification of distemper as an underlying cause. This case
Lumbar Cisternal example highlights the importance of including distemper
on the differential diagnosis list for young adult dogs with
Protein (mg/dL) 31 21
neurologic signs, because of the chronic course of disease
Total RBC (cells/ 111 2 that can occasionally occur. Additional laboratory tests
µL) that might or might not have been helpful for diagnosis
Total nucleated 1 3 of distemper in this dog include comparison of serum
cells (cells/µL) antibody titers to CDV in the CSF and serum, and RT-PCR for
CDV on CSF. Neurologic signs were progressive in this dog
Neutrophils (%) 0 0
and largely unresponsive to anticonvulsant drugs, and so
Small mononu- 92 80 the prognosis was extremely poor.
clear cells (%)
164 SECTION 1  Viral Diseases
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Newbury S, Larson LJ, Schultz RD. Canine distemper virus. In:
Miller L, Hurley K, eds. Infectious disease management in animal shel-
ters. Ames, IA: Wiley-Blackwell; 2009:161-172.
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CHAPTER 16
Canine Herpesvirus Infection
Autumn P. Davidson
Overview of Canine Herpesvirus Infection
First Described: New York State, United States, 19651
Cause: Canine herpesvirus-1 (Family Herpesviridae)
Affected Hosts: Domestic and wild Canidae
Geographic Distribution: Worldwide
Mode of Transmission: Direct oronasal contact with infected
secretions, transplacental transmission
Major Clinical Signs: Abortion in bitches; upper respiratory
disease and keratitis in adult dogs; in neonates, inappe-
tence, failure to thrive, vocalization, tachypnea, diarrhea,
neurologic signs, abdominal pain, nasal discharge, weight
loss, mucosal petechiation, death
Differential Diagnoses: Bacterial sepsis, infections with
other viruses such as canine distemper virus and canine
parvovirus
Human Health Significance: Canine herpesvirus-1 does not
infect humans.
Etiology and Epidemiology
Canine herpesvirus (CHV-1) is an enveloped virus that belongs
to the family Herpesviridae (Figure 16-1). It has been reported
from the United States, Canada, Australia, Japan, England, and
Germany. CHV-1 was first recognized in the mid-1960s in asso-
ciation with a fatal disease in puppies.2 The virus is commonly
blamed for acute neonatal puppy death or failure to thrive,
sometimes termed the “fading puppy syndrome.” When con-
firmed as a cause of disease, untreated CHV-1 infection in neo-
nates can cause high (up to 100%) mortality among littermates.
The virus is temperature sensitive and prefers to replicate at
temperatures less than 37°C. It is not stable in the environment
and is readily inactivated by disinfectants. As with other herpes-
viral infections, recovery from disease is associated with lifelong
latent infection of the neural ganglia, with periodic reactivation
of shedding in association with stress or immunosuppression,
such as that which results from overcrowding and pregnancy.
CHV-1 infection has not been reported in cats.
Clinical Features
Signs and Their Pathogenesis
Transmission of CHV-1 can occur subsequent to close contact
with infectious vaginal fluids during whelping or with vulvar
or oronasal secretions in the postpartum period. Exposure of
a naïve bitch to CHV-1 during the last 3 weeks of gestation
166
results either in late-term abortion of a litter or neonatal death
within the first few weeks of life, because inadequate peripartu-
rient maternal antibodies exist to allow passive immunity to be
acquired by the neonates. Puppies born to a naïve bitch may also
become infected as a result of contact with other dogs that shed
the organism. The incubation period of CHV-1 disease is 6 to
10 days. The virus replicates in the epithelial cells of the orona-
sal and pharyngeal mucosa, the genital tract, and the regional
lymphatics. Replication and spread of the virus is facilitated
by the presence of a low body temperature (<38°C or 100°F),
which is normal for canine neonates.
Older (>3 to 5 weeks of age) puppies that are exposed to
CHV-1 may develop subclinical infection, or the course of dis-
ease is less severe, as a result of their ability to mount a febrile
response. Latent infection also may develop. Concerns have
been raised about latency and the possibility of late develop-
ment of neurologic signs.3
Clinical signs are more likely to occur in animals that are
hypothermic or immunosuppressed. Signs in the neonate are not
specific and include incessant vocalization, anorexia (with poor
weight gain), dyspnea, abdominal pain, incoordination, diar-
rhea, serous to hemorrhagic nasal discharge, and petechiation
of the mucous membranes. The mortality rate in litters infected
in utero or during birth can approach 100%, with deaths occur-
ring during the first few days to a week of life. Exposed, surviv-
ing older neonates may develop a late onset of central nervous
signs including blindness, ataxia, and deafness; apparent com-
plete recovery has also been reported.
The recently infected brood bitch generally shows no clini-
cal signs. Healthy adult dogs of either gender can develop mild
upper respiratory signs (sneezing, serous oculonasal discharge,
keratitis) for a few days but are otherwise usually clinically
unaffected.4-6 Additional information on respiratory and ocular
disease caused by CHV-1 can be found in Chapter 17.
Diagnosis
Antemortem diagnosis of CHV-1 infection in neonates can
be challenging. Necropsy of a deceased littermate is usually
required.
Virus Isolation and Molecular Diagnosis Using
the Polymerase Chain Reaction
Tissues obtained at necropsy can be submitted for virus isolation
or molecular diagnosis using the PCR. Diagnosis by virus isola-
tion takes days. CHV-1–specific PCR assays can also confirm
infection, in a more timely fashion. Clinicians should confirm
the availability and turnaround time of commercially available
diagnostic tests when attempting to investigate a possible out-
break of CHV-1 infection in puppies.7-9
 CHAPTER 16  Canine Herpesvirus Infection 167

Pathologic Findings
Gross Pathologic Findings
Gross changes in the kidneys at necropsy of pups infected with
CHV-1 include multifocal petechial to ecchymotic subcapsular
hemorrhages (Figure 16-2). The pleural surfaces may be mottled
pink and red due to coagulation necrosis (Figure 16-2, B). Mul-
tifocal random acute necrosis of other organs, including the liver,
pancreas, intestine and adrenal glands, may also be apparent.
Lesions may resemble those of bacterial sepsis.
Histopathologic Findings
Histopathology can be used to confirm CHV-1 infection but may
be unrewarding in the clinical scenario unless very timely. Charac-
teristic viral intranuclear inclusion bodies can be difficult to find.
Histopathologic evaluation commonly reveals severe nephrosis
(proximal renal tubular epithelial necrosis and renal pelvic epithelial
apoptosis with swollen vesicular nuclei); diffuse severe hepatic con-
gestion and inflammation with vacuolar hepatopathy (neutrophilic,
histiocytic, and lymphocytic infiltration with apoptosis); acute
severe necrotizing bronchointerstitial pneumonia with pulmonary
edema and hemorrhage (terminal bronchiolar hemorrhage and
edema with fibrin accumulation, macrophage infiltration, necrosis,
and alveolar disruption); and thymic, lymph node, and splenic lym-
pholysis (severe diffuse cortical lympholysis with sinus congestion).

FIGURE 16-1  Structure of a herpesvirus. Herpesviruses are large, enveloped DNA
viruses. The DNA is contained within an icosahedral capsid.
Treatment
Treatment of puppies with CHV-1 infection is unrewarding and
rarely effective, and residual cardiac and neurologic damage
can occur in recovered animals. Treatment with immune serum
from seroconverted dams is typically ineffective in sick puppies,
and warming also has little effect.

C D
FIGURE 16-2  A, Kidneys from 12-day-old mastiff puppy from a litter of 17 puppies, 14 of which died. CHV-1 was isolated from the puppy. Gross changes include subcapsular hemor-
rhagic foci typical of (but not pathognomonic for) herpetic nephritis. On cut section, white and red cortical streaks are evident; a hemorrhagic medulla is evident. B, Multifocal pink and red
pleural mottling in the puppy described in A. Serosanguineous pleural fluid was present. Histopathology identified multiple areas of pulmonary necrosis and bronchopneumonia. C, Small-
intestinal serosal petechiation in the same puppy. Histopathology identified enterocyte necrosis consistent with CHV-1 infection. D, Liver of the same puppy showing uniform firmness
and diffuse mottle with rounded edges. Histopathology identified scattered multiple foci of acute hepatic coagulation necrosis; eosinophilic intranuclear inclusions typical of herpes viral
inclusions were seen within viable hepatocytes. (Images courtesy JR Peauroi.)
168 SECTION 1  Viral Diseases
An incompletely developed immune system and inadequate
thermoregulation during the first days of life means neonates
are vulnerable to systemic infection (by both bacterial and viral
pathogens). The umbilicus of neonates should be treated with
tincture of iodine immediately after birth to reduce contami-
nation and prevent bacterial ascent into the peritoneal cavity
(omphalitis-peritonitis). Neonatal bacterial peritonitis with bac-
teremia can cause rapid neonatal death if not recognized and
treated promptly (see Figure 34-1). Factors that predispose pup-
pies to bacteremia and sepsis include endometritis in the bitch,
a prolonged delivery/dystocia, feeding of replacement formulas,
the use of ampicillin, stress, low birth weight (<350 g), and chill-
ing with body temperatures less than 35.5°C or 95°F. Bacterial
organisms most frequently associated with bacteremia are Esche-
richia coli, streptococci, staphylococci, and Klebsiella spp. Com-
monly, a decrease in weight gain, failure to suckle, hematuria,
persistent diarrhea, unusual vocalization, abdominal distention
and pain, and sloughing of the extremities indicate sepsis may
be present. These signs are similar to those encountered with
CHV-1 infection. Prompt therapy with broad-spectrum, bacte-
ricidal antibiotics; optimal nutrition via supported nursing, tube
feeding, or bottle feeding; maintenance of body temperature; and
appropriate fluid replacement are indicated. The third-generation
cephalosporin ceftiofur sodium is an appropriate choice for treat-
ment of neonatal sepsis, because it alters normal intestinal flora
minimally and is usually active against the causative organisms.
Failure of neonatal sepsis to respond to antibacterial drug treat-
ment should prompt consideration of CHV-1 infection. Specific anti-
viral therapy, if instituted in a timely manner, may reduce mortality
from CHV-1. Acyclovir has activity against herpes simplex virus
(see Chapter 7). Its use in veterinary medicine is not well established,
and it should be used with caution and only in situations where
indicated. The safety and effectiveness in humans less than 2 weeks
of age is not established. The dose (20 mg/kg PO q6h for 5 days)
is extrapolated from that for humans.10 The efficacy of famciclovir
for treatment of CHV-1 infection requires study, but the pharmaco-
kinetics of famciclovir in dogs resembles that in humans,11 so much
lower doses may be required when compared with cats.
CASE EXAMPLE
Two, 25-day-old, female Labrador retriever puppies were exam-
ined because of acute tachypnea and vocalization. The puppies
belonged to a litter of eight. Whelping occurred without inci-
dent, and the pups showed normal weight gain and behavior
until the last 24 hours. Four days previously, the litter was vac-
cinated with an intranasal Bordetella bronchiseptica vaccine and
dewormed with pyrantel pamoate according to routine preven-
tative health care practice. The litter nursed from birth, and the
dam was normal.
Physical examination of both puppies revealed hypo-
thermia (rectal temperature 36.3°C [97°F] and 36.0°C [97°F],
respectively), tachypnea, a markedly tense abdomen, and
continuous vocalization. Despite supportive care (exogenous
warming, supplemental feeding with artificial bitch’s milk [Esbi-
lack Pet Ag, Inc.], subcutaneous lactated Ringer’s solution, and
Immunity, Vaccination, and Prevention
Minimal transplacental transfer of immunity occurs in the dog.
Adequate ingestion of colostrum must occur promptly postpar-
tum for puppies to acquire passive immunity. The transmission
of protective passive immunity (placental or colostral antibodies)
between a bitch and her puppies depends on the prior existence
of adequate serum maternal antibodies. Therefore, breeding
bitches with exposure to CHV-1 earlier in life have the best
opportunity to seroconvert and develop protective antibodies.
This commonly occurs in kennels or at crowded canine events
such as dog shows and trials. Documentation of positive CHV-1
serology generally indicates the presence of adequate maternal
antibodies.
Bitches who are naïve to CHV-1 during pregnancy must be
strictly isolated from potential exposure during gestation and
for at least 6 weeks postpartum to prevent transmission of the
virus to her fetuses or neonates. Subsequent litters of the infected
pregnant or postpartum bitch are usually normal because of
acquisition of maternal antibodies.
Vaccine development has been hampered by the poor immu-
nogenicity characteristic of the herpesviral vaccines devel-
oped for other species, such as feline herpesvirus-1 vaccines.
In Europe, a vaccine (Eurican Herpes 205) has been available
since 2003. Administration of two doses of the vaccine is rec-
ommended, first during estrus or early pregnancy and again 1
to 2 weeks before whelping. No suggestion for an evaluation
of the bitch’s serologic status prior to vaccination is made by
the manufacturer. Revaccination with two doses of the vaccine
is recommended at each pregnancy. No independent, nonpro-
prietary studies exist corroborating this vaccine’s efficacy or
benefit. The vaccine is not currently available in the United
States.12,13
Public Health Aspects
In general, herpesviruses are very host specific, and CHV-1 is
not known to infect humans.
ceftiofur sodium [2.5 mg/kg SC q12h]), the puppies died within
12 hours and then underwent necropsy examination. Tissues
collected for histopathologic evaluation included kidney, liver,
lung, thymus, mesenteric lymph node, and spleen.
Because of gross findings of hemorrhagic foci in the lungs
and kidneys, renal tissue was submitted for a CHV-1–specific
PCR assay. The PCR assay later confirmed the presence of CHV-1
DNA in the renal tissue. Gross (foci of hemorrhage in the lungs
and kidneys) and histopathologic evaluations of the deceased
puppies were typical of CHV-1 but were not confirmatory
because of the similarity of findings to those of neonatal bac-
terial sepsis. Intranuclear inclusion bodies were suspected
in macrophages from the lung of one puppy. Aerobic bacte-
rial culture of the peritoneal cavity was negative for bacterial
growth.
Poor weight gain and vocalization were reported in two
additional puppies. The primary differentials included neona-
tal bacterial sepsis and CHV-1 infection. Because of the lack of

response to therapy for bacterial infection in the two deceased
puppies, the remaining six puppies were treated with acyclovir
suspension (20 mg/kg PO q6h) for 7 days, and exogenous warm-
ing adequate to raise the rectal temperature to just above 37.7°C
(100°F). Therapy was initiated the day before the PCR assay con-
firmed infection with CHV-1.
All six treated puppies survived and were successfully
weaned between 5 and 6 weeks of age. All were placed in pri-
vate homes and had uneventful subsequent health histories
and normal behavior. Physical examinations including com-
plete fundic evaluations were performed at 12 to 14 months of
REFERENCES
1. Carmichael LE, Squire RA, Krook L. Clinical and pathological
features of a fatal viral disease of newborn pups. Am J Vet Res.
1965;26:803-814.
2. Carmichael LE, Greene CE. Canine herpesvirus infection. In:
Greene CE, ed. Clinical Microbiology and Infectious Diseases of
the Dog and Cat. Philadelphia, PA: WB Saunders; 1990:252-258.
3. Carmichael LE. Herpesvirus canis: aspects of pathogenesis and
immune response. J Am Vet Med Assoc. 1976;156:1714-1725.
4. Johnson SD, Root Kustritz MV, Olson PN. The neonate—from
birth to weaning. Canine and Feline Theriogenology. Philadelphia,
PA: WB Saunders; 2001:146-167.
5. Carmichael L. Neonatal viral infections of pups: canine herpesvirus
and minute virus of canines (canine parvovirus-1). In: Carmichael
L, ed. Recent Advances in Canine Infectious Diseases. Ithaca, NY:
International Veterinary Information Service (www.Ivis.org); 1999.
6. Percy DH, Carmichael LE, Albert DM, et al. Lesions in puppies sur-
viving infections with canine herpesvirus. Vet Pathol. 1971;8:37-53.
7. Evermann JF. Diagnosis of canine herpetic infections. In: Kirk RW,
Bonagura JD, eds. Current Veterinary Therapy. Philadelphia, PA:
WB Saunders; 1989:1313-1316.
CHAPTER 16  Canine Herpesvirus Infection 169
age and were normal in all. Two of the dogs have become active
guide dogs for the blind, and one is an assistance dog for the
hearing impaired. All remain physically normal at more than 26
months of age.
Comments: The epizootiology of this outbreak suggests that
the puppies were born from a naïve bitch and exposed to CHV-
1 from another carrier dog at days 14 to 17 after birth. Two of 8
(25%) succumbed to the disease; 2 of the remaining 6 showed
early clinical signs, after which antiviral therapy was initiated
for all dogs.
8. Burr PD, Campbell ME, Nicolson L, et al. Detection of canine her-
pesvirus 1 in a wide range of tissues using the polymerase chain
reaction. Vet Microbiol. 1996;53:227-237.
9. Decaro N, Amorisco F, Desario C, et al. Development and valida-
tion of a real-time PCR assay for specific and sensitive detection of
canid herpesvirus 1. J Virol Methods. 2010;169:176-180.
10. Plumb DC, ed. Plumb’s Veterinary Drug Handbook. Pocket Size.
6th ed. Hoboken, NJ: Wiley-Blackwell; 2008:18-19.
11. Filer CW, Ramji JV, Allen GD, et al. Metabolic and pharmacoki-
netic studies following oral administration of famciclovir to the cat
and dog. Xenobiotica. 1995;25:477-490.
12. Malone EK, Ledbetter EC, Rassnick KM, et  al. Disseminated
canine herpesvirus-1 infection in an immunocompromised adult
dog. J Vet Intern Med. 2010;24(4):965-968.
13. Verstegen J, Dhaliwal G, Verstegen-Onclin K. Canine and feline
pregnancy loss due to viral and non-infectious causes: a review.
Theriogenology. 2008;70(3):304-319.
CHAPTER 17
Canine Viral Respiratory Infections
Jane E. Sykes
Overview of the Canine Viral Respiratory
Infections
First Described: Respiratory disease in dogs (due to canine
adenovirus-2) was first described in 1961 in Canada.1 The
most newly recognized virus, canine pneumovirus, was
described in the United States in 2010.2
Proposed Causes: Canine adenovirus, canine influenza virus,
parainfluenza virus, canine respiratory coronavirus, canine
herpesvirus, possibly reoviruses and canine pneumovirus
Geographic Distribution: Worldwide
Mode of Transmission: Aerosol transmission or close contact,
sometimes fomites
Major Clinical Signs: Harsh or honking cough, serous nasal
discharge, conjunctivitis, fever (uncomplicated disease).
Fever, lethargy, inappetence, tachypnea, productive
cough, mucopurulent nasal and ocular discharge, rarely
death (complicated disease).
Differential Diagnoses: Upper and lower respiratory diseases
such as airway collapse, bordetellosis, Streptococcus equi
subsp. zooepidemicus infection, fungal or protozoal pneu-
monia, respiratory tract neoplasia, airway foreign bodies,
chronic bronchitis, eosinophilic bronchopneumopathy
or granulomatosis, parasitic infections (such as Filaroides,
Oslerus, Capillaria, Paragonimus, Dirofilaria), aspiration
bronchopneumonia, left-sided congestive heart failure
Human Health Significance: Viruses that cause canine infec-
tious respiratory disease generally are not considered
zoonotic. Canine parainfluenza and reoviruses repre-
sent potential zoonoses, but the significance of this is
unknown.
Etiology and Epidemiology
Canine viral respiratory disease is a widespread problem where
large numbers of dogs are housed indoors together, such as
in shelters, commercial dog colonies, and breeding facilities.
The longer dogs are housed in a shelter situation, the greater
the risk that respiratory illness will occur.3 In shelter environ-
ments, transmissible respiratory disease (or canine infectious
respiratory disease complex [CIRDC], previously referred to as
“­kennel cough” or canine infectious tracheobronchitis) delays
the placement of dogs in homes and can result in unmanageable
costs related to treatment, quarantine, and isolation. Trans-
missible respiratory disease occasionally occurs in owned dogs
170
after a period of contact with large numbers of other dogs at
dog parks, dog sporting events such as fly ball, or dog behavior
classes. It can also occur after dogs (or dog owners) visit veteri-
nary hospitals, boarding facilities, or pet daycare centers.
With the widespread clinical application of molecular diag-
nostic assays, it is increasingly apparent that the number of
viruses that can infect the canine respiratory tract is much
larger than previously thought. This has led to exciting new
discoveries in the field of canine infectious respiratory disease,
and our knowledge of the pathogens involved continues to
expand. Co-infections with multiple viruses and bacteria such
as Mycoplasma spp., Bordetella bronchiseptica, and Strepto-
coccus equi subspecies zooepidemicus are common and con-
tribute to an increased severity of disease.4-6 Viruses believed
to play a role in canine respiratory disease include canine her-
pesvirus-1 (CHV-1), canine adenovirus-2 (CAV-2), canine dis-
temper virus (CDV), canine parainfluenza virus (CPiV), canine
pneumovirus, canine respiratory coronavirus (CRCoV), and
canine influenza virus (CIV) (Table 17-1). CAV-1 may also
play a role when vaccination is not performed or when it is per-
formed improperly.7 Reoviruses may also play a role.8 CDV,
CHV-1, and CAV are discussed in more detail in Chapters 15,
16, and 18, respectively. The major bacterial causes of canine
transmissible respiratory disease are covered in Part II, Section
2 of this book (Chapters 34, 38, and 40). Although most of
the canine respiratory viruses have a worldwide distribution,
their relative prevalence varies from year to year and between
geographic locations. Even within a state or city, predominant
pathogens may differ from one shelter and boarding kennel to
another.
CDV, CHV-1, CPiV, CRCoV, and CIV are enveloped
viruses, so they survive poorly in the environment and are sus-
ceptible to a variety of disinfectants. Despite this, contact with
virus that persists in the environment may be important for
transmission in densely housed populations of dogs. CAV-2 is
a non-enveloped virus and has the potential to survive several
weeks on fomites.
Canine Adenovirus-2
Adenoviruses are icosahedral DNA viruses that infect a vari-
ety of animal species. CAV-2 is found worldwide and primarily
infects the respiratory tract of dogs. Rarely, it has been impli-
cated as a cause of enteritis in dogs, and it was found in the
brains of puppies with neurologic signs.9 The virus replicates
in nonciliated bronchiolar epithelial cells; epithelial cells of the
nasal mucosa, pharynx, and tonsillar crypts; mucous cells in the
trachea and bronchi; and type 2 alveolar epithelial cells. CAV-2
can also be isolated from retropharyngeal and bronchial lymph
nodes as well as epithelial cells of the intestinal tract. Shedding
typically ceases 1 to 2 weeks after initial infection.
 CHAPTER 17  Canine Viral Respiratory Infections
TABLE 17-1
Potential Causes of Transmissible Respiratory Disease in Dogs
Organism Incubation Period (days)*
Canine adenovirus-1 4 to 9
Canine adenovirus-2 3 to 6
Canine distemper virus 3 to 6, longer for neurological signs
Canine herpesvirus-1 6 to 10; may be longer with
­stress-induced reactivation
Canine influenza virus 2 to 4
Canine parainfluenza virus 3 to 10
Canine respiratory coronavirus Probably days
Bordetella bronchiseptica 2 to 6
Mycoplasma cynos 3 to 10
Streptococcus equi subspecies Probably days
zooepidemicus
*Incubation and shedding periods are approximate and may differ when co-infections or immunosuppression operate.
Canine Herpesvirus-1
The extent to which CHV-1 plays a role in respiratory disease
in dogs has been debated.10 Experimental infections of dogs can
result in rhinitis or signs of tracheobronchitis, and intraocular
infection results in conjunctivitis and keratitis.11-14 There is evi-
dence of widespread exposure to CHV-1 in dogs worldwide,10 and
a substantial proportion of the dog population may be latently
infected. Like other herpesviruses, CHV-1 becomes latent in
neurologic tissues, with precipitation of virus shedding by stress.
Consistent with the time delay required for reactivation after
stress, CHV-1 was most frequently detected 3 to 4 weeks after
dogs were introduced to a rehoming center, whereas CRCoV and
CPiV were most frequently detected in the first and second week.
Dogs infected with CHV-1 were more likely to have severe respi-
ratory disease, although severe respiratory disease itself might
predispose dogs to the shedding of CHV-1.10 Infection with
CHV-1 was reported in association with fatal hepatic necrosis in
an adult dog that lacked any evidence of immunosuppression.15
Refer to Chapter 16 for information on CHV-1 infections in
neonates.
Canine Influenza Virus
Influenza viruses are enveloped viruses with segmented single-
stranded RNA genomes that belong to the family Orthomyxo-
viridae. Influenza viruses that cause disease in domestic animals
belong to the genus Influenzavirus A, whereas influenza B and
influenza C viruses primarily circulate among humans. Influenza
A viruses are classified based on the genetic composition of their
hemagglutinin (H) and neuraminidase (N) genes. To date, 16
H types and 9 N types have been identified, each of which are
antigenically distinct.16 The names of influenza viruses are spec-
ified as follows: influenza genus (A, B, or C)/host/geographic
origin/strain number/year of isolation and, in parentheses, H
and N type—for example, A/canine/Florida/43/2004 (H3N8).17
Extensive genomic rearrangements that occur within influenza
A viruses allow for occasional cross-species transmission among
171
Shedding Period* Environmental Survival
6 to 9 months Weeks to months
1 to 2 weeks Weeks to months
Weeks to months Hours
Unknown Hours
7 to 10 days Hours
8 to 10 days Hours
6 to 8 days Hours
Months Has the potential survive and
grow in environmental water
for several weeks
Months Hours
At least 2 weeks Unknown; other streptococci
can survive weeks
birds and mammals. These rearrangements occur when two
different viruses simultaneously infect a host, with subsequent
genetic reassortment. Occasionally, cross-species transmission
occurs without alteration of the viral genome.
In the United States, CIV emerged in racing greyhounds in
Florida in 2003 and 2004,17 where it caused hemorrhagic pneu-
monia and a high mortality. However, serologic evidence of
infection in the greyhound dog population dates back to 1999.18
Infections spread slowly and were subsequently reported in rac-
ing greyhounds and non-greyhounds in at least 38 U.S. states.
Outbreaks have continued to occur in shelter situations for nearly
a decade after the virus was discovered. The virus that currently
circulates in the United States is an H3N8 virus that closely
resembles an equine influenza virus, which suggested that an
interspecies jump occurred without genetic reassortment.17
Instead, accumulation of point mutations with minor amino
acid changes occurred, with sustained transmission among
dogs. The most significant outbreaks of disease due to CIV have
occurred in Florida, New England, Colorado, Wyoming, and
Texas. In many other states, sustained transmission of the virus
from one dog to another has not occurred. The most significant
risk factor for infection has been indoor housing.19 Virtually all
cases to date have involved dogs in kennels, animal shelters, or
dog daycare facilities. Dogs of all ages and breeds are suscep-
tible, but to date severe hemorrhagic pneumonia has occurred
only in greyhounds.20 The virus is shed for up to 7 to 10 days.
CIV has retained the ability to infect horses, but horses develop
only mild disease or no clinical signs.20
Avian-lineage H3N2 CIV emerged in South Korean dogs
in 2007, and a similar virus was subsequently isolated from
dogs in China.21,22 Experimentally, cats are also susceptible
to infection by this virus.23 Korean isolates were associated
with epidemics of respiratory disease in kennels within veteri-
nary clinics.22 A novel H3N1 virus was also detected in South
Korean dogs that lacked signs of respiratory disease.24 A novel
H5N2 influenza virus was recently detected in a dog with
172 SECTION 1  Viral Diseases
respiratory disease in China.25 Dogs are susceptible to infection
with human influenza virus H1N126,27 and avian H5N1,28 but
sustained transmission of these viruses in the dog population
has not been reported. Limited infection of dogs with equine
H3N8 viruses was detected in hounds in England29 and during
an equine influenza outbreak in Australia.30 In England, disease
was so severe that several hounds had to be euthanized, and
subacute bronchointerstitial pneumonia was detected at nec-
ropsy.29 The Australian dogs developed inappetence, lethargy,
nasal discharge, and a cough that persisted for several weeks,
but dog-to-dog transmission was not identified. Experimental
transmission of H3N8 influenza virus from horses to dogs was
documented in Japan, but infected dogs did not show signs of
illness.31
Canine Parainfluenza Viruses
CDV, CPiV, and canine pneumovirus belong to the family
­Paramyxoviridae, which are enveloped RNA viruses. CPiV
belongs to the genus Rubulavirus. Although previously referred
to as canine parainfluenza virus-2, it is probably the same virus
as simian virus 5, which was originally isolated from monkey
cell cultures. It has been proposed that it be renamed parainflu-
enza virus 5.32 Unlike CDV, the outer envelope possesses not
only hemagglutinin but also neuraminidase activity (HN attach-
ment glycoprotein). The virus infects dogs worldwide, replicates
in epithelial cells of the upper respiratory tract, and often causes
no signs or mild respiratory illness. Respiratory disease may be
more severe when co-infections with other pathogens such as
B. bronchiseptica are present.33 Viremia seems to be uncom-
mon, but occasionally CPiV has been isolated from the liver,
spleen, and kidneys. A strain of CPiV was also isolated from a
dog with neurologic signs.34 There is some evidence that cats
may be infected with CPiV or a closely related virus.35 Virus is
shed for up to 10 days after infection.
Canine pneumovirus belongs to the genus Pneumovirus. It
was first isolated from dogs with acute respiratory disease in
shelters in the United States in 2010.2 The virus is most closely
related to a murine pneumovirus and in fact can replicate in
mice and cause severe inflammatory pathology.36
Canine Reoviruses
Reoviruses (family Reoviridae, genus Orthoreovirus) are non-
enveloped viruses with a segmented, double-stranded RNA
genome. Mammalian reoviruses infect a variety of host species
and have a worldwide distribution. The prefix reo- stands for
respiratory enteric orphan virus, which highlights the tropism of
reoviruses for cells of the respiratory and gastrointestinal tract,
and their uncommon association with disease. Despite serologic
evidence of widespread exposure to reoviruses in dogs, they
have been found only rarely in dogs with respiratory disease8,37
and dogs with enteritis.38 There are three mammalian reovi-
rus (MRV) serotypes, and all three have been detected in dogs.
The role of reoviruses in disease causation in dogs is unclear,
because disease has not been reproducible experimentally. It has
been speculated that reoviruses might act synergistically with
other respiratory pathogens to cause disease.10
Canine Respiratory Coronavirus
Coronaviruses are enveloped RNA viruses that possess large,
club-shaped spikes on their outer surface, also known as pep-
lomers (see Figure 14-1, B). CRCoV is a relatively newly identi-
fied cause of contagious respiratory disease in dogs. The virus is a
BOX 17-1
Genetic Relationships between Coronaviruses
Group 1a
Feline coronavirus
Canine enteric coronavirus
Transmissible gastroenteritis virus of swine
Porcine respiratory coronavirus
Ferret and mink coronaviruses
Group 1b
Bat coronavirus HKU8
Human coronavirus 229E
Human coronavirus NL63
Porcine epidemic diarrhea virus
Group 2a
Canine respiratory coronavirus
Bovine coronavirus
Human coronavirus OC43
Human coronavirus HKU3
Mouse hepatitis virus
Porcine hemagglutinating encephalomyelitis virus
Group 2b
SARS coronavirus (humans)
Bat SARS coronavirus HKU3
Group 3
Avian infectious bronchitis virus
Turkey coronavirus
SARS, Severe acute respiratory syndrome
group 2a coronavirus (family Coronaviridae, genus ­Coronavirus)
and is distinct from canine enteric coronavirus, a group 1a coro-
navirus (Box 17-1).39 Minimal serologic cross-reactivity exists
between these two viruses. Group 2a coronaviruses possess a
gene that encodes hemagglutinin esterase, an outer membrane
protein glycoprotein. This gene is absent in group 1 and 3 coro-
naviruses. CRCoV is most closely related to a bovine coronavi-
rus but also resembles human coronavirus OC43.40
CRCoV was first reported in 2003, in a group of dogs with
respiratory disease in a rehoming facility in England that had
been vaccinated against CAV-2, CDV, and CPiV.6,40 Some of
the dogs were co-infected with CPiV and CHV-1.6 CRCoV
spread rapidly and primarily was detected in the first week that
dogs were introduced to the kennel, after which time CPiV and
CHV-1 were detected. Alone, CRCoV causes subclinical infec-
tions or mild respiratory disease, but like human respiratory
coronaviruses, it can cause reversible damage to, or loss of, the
cilia on respiratory epithelial cells (Figure 17-1). As a result,
infected dogs are predisposed to secondary infections. Serologic
evidence of exposure to CRCoV is widespread in dogs from
North America, Great Britain and continental Europe, Japan,
Korea, and New Zealand,10,41-43 and the virus has been detected
widely using PCR-based methods in dogs with respiratory dis-
ease from many of these countries. To date, there is no evidence
that cats can be infected with CRCoV.
 CHAPTER 17  Canine Viral Respiratory Infections
A B
FIGURE 17-1  Effect of canine respiratory coronavirus infection on ciliated respiratory
epithelial cells. A, Respiratory epithelium immediately before inoculation with CRCoV. B,
The same cells 48 hours after inoculation with CRCoV. There is marked loss of cilia, thinning
of the epithelium with individual cell necrosis, and sloughed necrotic cells on the surface.
There are also occasional large vacuoles within the epithelium, which likely represent foci
of cell loss secondary to viral replication. (Courtesy Dr. Simon Priestnall and Professor Joe
Brownlie, Royal Veterinary College, UK.)
Infection with pancytotropic strains of canine enteric coro-
navirus has recently been associated with severe systemic dis-
ease in puppies from Europe. Affected pups have fever, mucoid
to hemorrhagic enteritis, lymphopenia, neurologic signs such
as seizures, and bronchopneumonia.44,45 Co-infections with
CPV-2 were present in some puppies, but the disease has been
reproduced by experimental infection by canine enteric corona-
virus alone. Death occurred in some puppies, but others devel-
oped transient gastrointestinal signs and recovered.46
Clinical Features
Signs and Their Pathogenesis
The incubation period for viral respiratory disease is generally
less than 2 weeks and can be as short as 2 to 3 days. Influ-
enza virus infections, in particular, have been associated with
very short incubation periods.20 Transmission occurs by aero-
sol, but direct contact between dogs and fomite transmission
(hands, clothing, contaminated food and water bowls, common
hallways and exercise areas) are important routes in popula-
tion-dense environments. Cells of the larynx, trachea, bronchi,
and sometimes the nasal mucosa, bronchioles, and alveoli are
infected, and although viral shedding patterns differ between
pathogens, shedding may occur before the onset of clinical
signs. The duration of shedding for CIV and CPiV is very short,
typically a few days, and in some dogs, shedding has ceased by
the time clinical signs are most apparent. In contrast, CDV can
be shed for weeks (see Chapter 15).
Infection with respiratory viruses may be associated with
no signs, or complicated pneumonia and death can occur. In
general, morbidity is high, but mortality is low. Moderate to
severe signs may be more likely to occur in very young puppies,
genetically susceptible animals, and when stress and co-infec-
tions with multiple viral and bacterial pathogens are present.
Infection with CDV is especially effective at predisposing dogs
to other respiratory viral infections, because of its immuno-
suppressive properties, but other viruses that damage ciliated
epithelial cells, such as CRCoV and CPiV, also predispose to
co-infections. Clinical signs of acute respiratory disease include
173
A
B
FIGURE 17-2  A, Right eye of a 10-year-old male neutered miniature schnauzer
infected with bilateral conjunctivitis and dendritic ulceration associated with canine her-
pesvirus infection. Fluorescein uptake can be seen in a branching pattern at the limbus of
the right eye. B, The same dog 1 month later. Note diffuse stain uptake over at least 25%
of the corneal surface. (Courtesy University of California, Davis Veterinary Ophthalmology
Service.)
mild fever, a paroxysmal harsh or “honking” cough, serous
nasal discharge, and sometimes sneezing, but usually otherwise
affected dogs are often alert, active, and appetent. The cough
may be followed by gagging or retching, which may be followed
by the production of frothy mucus. An altered bark or stridor
may occur in dogs that develop laryngitis, tonsillitis, and/or
pharyngitis. CAV-2 and CHV-1 may cause conjunctivitis.47 In
addition, CHV-1 has been associated with ulcerative and non-
ulcerative keratitis, which may be precipitated by immunosup-
pression (Figure 17-2).48-50 Infections with CIV or CDV, or
infections with multiple respiratory pathogens, may be more
likely to produce systemic signs of fever and lethargy. Dogs that
develop secondary bacterial infections may show fever, inappe-
tence, lethargy, a mucopurulent nasal discharge, tachypnea, and
a moist, productive cough.
Physical Examination Findings
On physical examination, dogs with uncomplicated transmissible
respiratory disease are typically bright, alert, and active with a
paroxysmal cough. The cough is often easily elicited on tracheal
palpation. Conjunctivitis and serous ocular and/or nasal dis-
charge may be present, and the tonsils may be enlarged and hyper-
emic. Dogs with secondary bacterial infections may be pyrexic,
lethargic, and tachypneic with increased respiratory effort and
174 SECTION 1  Viral Diseases

TABLE 17-2
Diagnostic Assays Available for Respiratory Viruses in Dogs
Assay Specimen Type Target Performance
Virus isolation Conjunctival, nasal, and caudal Virus False negatives can occur in specimens that contain
pharyngeal swabs; transtracheal no or low numbers of virus particles. Attenuated
and bronchoalveolar wash speci- live vaccine virus may also be isolated. May take
mens; airway and lung speci- several days and requires specialized techniques
mens collected at necropsy and expertise.
Influenza ELISA Same as virus isolation CIV antigen Inexpensive and rapid. Sensitivity and specific-
antigen assay ity in naturally infected dogs have not been well
established, so results should be interpreted with
caution.
PCR or RT-PCR Same as virus isolation Viral nucleic acid Sensitivity and specificity may vary with assay design.
Sensitivity may be low because of brief shedding
for some viruses. Assays may not be available for
all viruses or may not detect all virus strains. Test-
ing specimens from multiple different anatomic
sites or combining PCR with other diagnostic tests
can increase sensitivity. Attenuated live vaccine
virus may be detected after vaccination. Because of
subclinical shedding, the significance of a positive
result may be difficult to interpret. False-negatives
may occur as a result from degradation of viral
nucleic acid during specimen transport.
Serology Serum Antibodies against Interpretation complicated by previous vaccination
respiratory viral and exposure. May be useful when the exposed
antigens population is naïve and unvaccinated for the virus
of interest. Acute and convalescent sera required
for diagnosis.
Histopathology Necropsy specimens Viral inclusions; Can be used for diagnosis at necropsy. Detection of
immunostaining inclusions has low sensitivity, and immunostaining
and/or PCR is required to definitively identify the
infecting virus.

CIV, Canine influenza virus; RT, reverse transcriptase.

increased lung sounds on thoracic auscultation. A mucopurulent
nasal discharge may also be present. Ulcerative or nonulcerative
keratitis may be present in dogs infected with CHV-1.
Diagnosis
It is not possible to identify the cause of transmissible respi-
ratory disease in dogs based on clinical signs alone, because
each pathogen produces a similar spectrum of signs. The high
prevalence of co-infections further complicates diagnosis. In
addition, other, noncontagious causes of respiratory disease in
dogs can lead to signs that closely resemble transmissible respi-
ratory disease. A history of exposure to other dogs can support
the diagnosis.
Most dogs experience self-limiting disease. Attempts to obtain
an etiologic diagnosis should be made when disease persists for
longer than 7 to 10 days or is complicated by bacterial pneumo-
nia, with lethargy and inappetence. When outbreaks occur in
shelters or the pattern of endemic respiratory disease changes,
attempts to make an etiologic diagnosis are also indicated and
encouraged (Table 17-2). Collection of multiple specimen types
from several dogs with and without clinical signs in an outbreak
situation can facilitate diagnosis and allow interpretation of the
significance of positive test results. Organism detection meth-
ods, such as PCR assays, are likely to be of highest yield early
in the course of illness (e.g., the first 1 to 3 days) or in exposed
dogs that have not yet developed clinical signs. Use of a combi-
nation of serology and organism detection methods also facili-
tates diagnosis. In shelter situations or outbreaks where severe
disease occurs, necropsies can provide valuable information and
should be performed by a veterinary pathologist as soon as pos-
sible after death or euthanasia. Tissues should be submitted for
histopathology (in formalin), bacterial and virus cultures (fresh
tissue), and PCR assay for respiratory viruses and bacteria (fresh
or frozen tissue; see Chapter 5).
Laboratory Abnormalities
There are no specific abnormalities in the CBC, serum biochem-
istry profile, or urinalysis that aid in a diagnosis of canine viral
respiratory disease. The CBC may be normal or show a mild to
moderate neutrophilia. Band neutrophils and neutrophil toxic-
ity may be apparent in dogs with secondary bacterial pneumo-
nia. Severe infections may be associated with leukopenia, which
may be followed by leukocytosis in dogs that recover.
 CHAPTER 17  Canine Viral Respiratory Infections
FIGURE 17-3  Dorsoventral thoracic radiograph from a 2-year-old male neutered
coonhound that had left a boarding facility 6 days previously with a moist, productive
cough that subsequently progressed and was complicated by the development of fever
and a mucopurulent nasal discharge. A CBC showed neutrophilia (17,840 cells/µL),
increased numbers of band neutrophils (223 cells/µL), and monocytosis (3,345 cells/µL).
Bronchopneumonia is present that involves the right cranial, right middle, and left cranial
lung lobes. Interestingly, canine herpesvirus DNA was detected in a pooled extract from
nasal, conjunctival, and oral swab specimens, as well as a whole blood sample using a
PCR assay.
Transtracheal wash and bronchoalveolar lavage specimens
from dogs with viral pneumonia typically show a suppurative or
mixed exudate, sometimes with evidence of intracellular bacteria.
Culture for aerobic bacterial culture and Mycoplasma spp. are
indicated on wash specimens, and antimicrobial susceptibilities
should be obtained for any aerobic bacteria isolated. Organisms
such as Pasteurella spp., Staphylococcus pseudintermedius, Strep-
tococcus canis, Escherichia coli, Klebsiella pneumoniae, and some
Mycoplasma spp. infect the airways as opportunists and are not
considered to be primary pathogens. Bacterial culture of nasal
swabs is generally not recommended because it often leads to
growth of normal flora, which has no clinical relevance.
Diagnostic Imaging
Plain Radiography
In uncomplicated viral infections, plain thoracic radiography
in dogs often shows no significant abnormalities, or there may
be a mild diffuse interstitial or bronchointerstitial pattern. Sec-
ondary bacterial bronchopneumonia may be characterized by
development of peribronchial and alveolar infiltrates or lobar
consolidation (Figure 17-3).
175
Microbiologic Tests
Virus Isolation
Efforts to isolate viruses from dogs with acute respiratory
disease have been most useful for identification of novel or
reemerging pathogens.2,38,40 Despite the increased availability
of molecular diagnostic assays, virus isolation is still offered to
veterinarians for routine diagnostic purposes by laboratories
that specialize in virology (e.g., the Animal Health Diagnos-
tic Laboratory at Cornell University in Ithaca, NY). Suitable
specimens for respiratory virus isolation are nasal and pharyn-
geal swab specimens, transtracheal or bronchoalveolar lavage
specimens, or upper airway or lung tissue obtained at nec-
ropsy. If swabs are used to collect specimens, polyester-tipped
swabs and specific virus transport media should be used. Cot-
ton swabs should be avoided, because influenza viruses adhere
to the cotton, which can lead to reduced sensitivity.20 Virus
isolation is a specialized process that may take several days.
As with PCR assays, sensitivity may be low because shedding
of respiratory viruses occurs early in the course of illness and
may be transient. CRCoV fails to replicate in many cell lines,
but was successfully isolated in HRT-18 cells.51 This may
explain its relatively recent discovery relative to other respira-
tory viral pathogens.
Fluorescent Antibody Testing
Fluorescent antibody can be applied to smears made from swabs
from the upper respiratory tract, or tissues collected at necropsy,
such as fresh lung tissue. Assays are available for detection of
CPiV, CAV, CDV (see Chapter 15), and CHV-1. Attenuated
live vaccination with intranasal vaccines for CPiV and CAV has
the potential to interfere with fluorescent antibody results, but
this requires further study. The sensitivity of fluorescent anti-
body testing is likely to be lower than that of PCR assays, and
inexperienced personnel may misinterpret nonspecific fluores-
cence as a positive result.
Enzyme-Linked Immunosorbent Assays for Influenza Virus Antigen
Point-of-care ELISAs are available for detection of nucleopro-
tein antigen of human influenza A viruses. The assays are easily
performed and provide rapid results. Unfortunately, such assays
have had limited sensitivity and specificity for diagnosis of CIV
infections, and so their use is not recommended. False positives
may especially be a problem in shelters where canine influenza
is not endemic.
Molecular Diagnosis Using the Polymerase Chain Reaction
Panels of real-time PCR assays that detect respiratory patho-
gens are offered by some commercial veterinary diagnostic
laboratories. These may include assays for CRCoV, parainflu-
enza virus, and CIV, as well as bacterial pathogens such as B.
bronchiseptica and Mycoplasma spp. Swabs of the nasal cav-
ity and/or caudal pharynx, or respiratory lavage specimens
could be submitted for testing. At necropsy, CRCoV is most
readily detected in upper respiratory tract specimens, such as
the nasal mucosa, nasal tonsil, and trachea.52 Lung specimens
may also yield positive results. Unfortunately, false-negative
PCR results are common, especially in antemortem specimens,
because of transient or low-level shedding of many respiratory
viruses. In addition, because many respiratory viruses are RNA
viruses and RNA is very labile when compared with DNA, false
negatives can occur when viral RNA degrades during specimen
transport.
176 SECTION 1  Viral Diseases
In outbreak situations, false-positive PCR results may occur
if swab specimens become contaminated with virus from the
environment or the hands of personnel. Clean examination
gloves should be worn for each dog, and the swab should only
touch the anatomic site to be tested. Vaccination with live
attenuated vaccines (especially intranasal vaccines) may cause
positive test results with real-time PCR assays, but the extent to
which this occurs in dogs with respiratory viral illness requires
further study. Because CIV vaccines are parenteral inactivated
vaccines, they would not be expected to lead to false-positive
PCR assay results. The detection of nucleic acid from a respira-
tory virus in a specimen may not imply disease causation.
Serologic Diagnosis
Diagnostic laboratories that specialize in veterinary virology
may offer serologic assays for antibodies to canine respiratory
viral pathogens on a commercial basis to practitioners. Serology
has limited use for diagnosis because of vaccine titer interference
for some organisms, and the high prevalence of subclinical expo-
sure to organisms endemic in the dog population. Titers may
be negative in the first 10 days of illness, and some dogs may
not show a significant increase in antibody titer after infection.
Despite these limitations, serologic assays have been key to iden-
tification of infection and disease caused by emerging pathogens
such as CIV, when the disease is not endemic.20 In this situation,
analysis of paired serum specimens collected 2 weeks apart can
be used to document recent infection. In some dogs, no other
diagnostic test may be useful for antemortem diagnosis because
virus shedding is so transient and difficult to detect.
Serologic assays for CIV exposure are based on serum neu-
tralization or hemagglutination-inhibition (see Chapter 2).
Assays for CIV that use equine influenza virus antigen for
antibody detection have suboptimal sensitivity, whereas an
assay based on H3N8 CIV antigen was sensitive and specific
when compared with serum neutralization.53 Seroconver-
sion to CRCoV in a bovine coronavirus antigen ELISA assay
helped identify the association between infection with this
virus and respiratory disease in dogs in the rehoming kennel
in the United Kingdom.6 Serum neutralization assays can be
performed for detection of antibodies to CAV-2, CPiV, and
CHV-1. Antibodies to CAV-2 and CPiV can also be detected by
hemagglutination-inhibition.
Pathologic Findings
Gross Pathologic Findings
Necropsy of dogs with viral respiratory infections may show
no gross lesions, or pulmonary consolidation and hyperemia of
the tracheal mucosa may be present. Purulent exudate may be
present in the bronchi. Greyhounds that died of CIV infection
had extensive hemorrhage within the lungs, mediastinum, and
pleural space, together with mild, fibrinous pleuritis.17
Histopathologic Findings
Histopathology of the airways of dogs with viral respiratory
infections may show loss or irregularity of normal respiratory
cilia and epithelial necrosis and ulceration within the trachea,
bronchi, and bronchioles. With secondary bacterial infection,
a neutrophilic or mixed inflammatory infiltrate may be seen
in the airway mucosa, submucosa, and alveoli.54 Bronchial
and bronchiolar lumina may contain neutrophils, macro-
phages, and cellular debris. Thickening of the alveolar walls
with type 2 pneumocyte hyperplasia and interstitial edema has
also been described. Hemorrhagic interstitial and bronchoint-
erstitial pneumonia, vasculitis, and thrombus formation have
been observed in greyhounds with CIV infection.20 CAV-2 can
produce large, basophilic intranuclear inclusion bodies within
bronchial, bronchiolar, and alveolar cells. These must be differ-
entiated from the eosinophilic inclusions of CDV (see Chapter
15).55 Reoviruses can also produce intracytoplasmic inclusions
within bronchial epithelial cells.8 Use of immunohistochemistry
can facilitate definitive identification of viral antigen within cells
of the respiratory tract.5,55
Treatment and Prognosis
Like the common cold in humans, contagious respiratory dis-
ease in dogs resolves without treatment in the vast majority of
dogs, regardless of the underlying cause. For dogs with signs
of respiratory disease that have been present for less than 7 to
10 days and that remain bright and appetent, no treatment is
indicated. In some dogs, cough persists for as long as 10 to 30
days. Cough suppressants such as hydrocodone could be used for
dogs with a nonproductive, honking cough that occurs through-
out the day and night. Cough suppressants should not be used in
dogs that have productive cough, because they suppress normal
clearing mechanisms. Use of a harness or gentle leader for leash
walking rather than a neck collar may also reduce cough. The
efficacy and optimal dosage of neuraminidase inhibitors such
as oseltamivir (see Chapter 7) is unknown, and because of this
and the fact that oseltamivir is a first-line treatment for pandemic
influenza in humans, it should not be used to treat dogs with
respiratory disease, even when CIV infection is present. For dogs
with confirmed ocular CHV-1 infections that are associated with
corneal ulceration, topical antiviral ophthalmic preparations such
as idoxuridine or cidofovir could be considered (see Chapter 7).50
Antimicrobial drug treatment could be considered for dogs
with signs that persist beyond 7 to 10 days, but is primarily
indicated when there is evidence of secondary bacterial bron-
chopneumonia, such as pulmonary alveolar infiltrates and con-
solidation on thoracic radiography, lethargy, mucopurulent
oculonasal discharges, and/or decreased appetite. Because anti-
microbial drug resistance is increasingly reported among sec-
ondary bacterial pathogens and B. bronchiseptica, treatment of
dogs with secondary bacterial bronchopneumonia is optimally
based on the results of culture of a transtracheal wash specimen
from each affected dog and antimicrobial susceptibility test-
ing. For dogs with severe pneumonia, initial treatment should
involve the use of broad-spectrum parenteral antimicrobial
drugs such as a combination of a fluoroquinolone and a peni-
cillin or clindamycin. When infection with B. bronchiseptica
or Mycoplasma spp. is suspected, doxycycline may be the best
first choice. Indiscriminate use of antimicrobial drugs leads only
to widespread bacterial resistance and failure of antimicrobial
drug treatment in dogs that develop severe disease.
Dogs with pneumonia may also require treatment with intra-
venous fluids, supplemental oxygen, nebulization, and coup-
age. Nutritional support in the form of feeding tubes may be
required for dogs that are inappetent.
Prognosis depends on the virulence of the causative agent(s),
the presence of co-infections, and other factors that contribute
to host immunosuppression. Prognosis is generally excellent for
dogs with uncomplicated infections with a single pathogen. For
CIV infection, mortality rates have been less than 8% and could
be lower with rapid diagnosis and appropriate treatment.20
 CHAPTER 17  Canine Viral Respiratory Infections
Immunity and Vaccination
Vaccines are available for reduction of disease due to CPiV,
CAV-2, CDV, and CIV. With the exception of CDV vaccines,
none of the available vaccines completely prevent infection and
shedding, but they can lessen the severity of disease, provided
other factors such as overcrowding and appropriate disinfec-
tion and reduction of other stressors are also addressed. Paren-
teral and mucosal (intranasal and oral) attenuated live vaccines
are available for CPiV and CAV-2. The vaccine for CIV is an
inactivated vaccine. Vaccines for canine transmissible respira-
tory disease are considered noncore vaccines, so they should
be administered to dogs at risk of exposure, such as those that
enter shelters, boarding kennels, shows, sporting competitions,
popular dog parks, or pet daycare facilities.
Canine Adenovirus-2
Mucosal and parenteral vaccines are available for prevention
of disease due to CAV-2. Parenteral CAV-2 vaccines also pro-
tect against CAV-1 infection. Maternal antibodies persist for up
to 12 to 14 weeks after birth. Mucosal vaccines may be useful
to overcome maternal antibodies in young dogs that are intro-
duced to shelter environments. However, parenteral vaccines
are still required for adequate protection against CAV-1.
Canine Herpesvirus
An inactivated vaccine for CHV-1 is available in Europe for
pregnant bitches to protect puppies against neonatal infections
(see Chapter 16). It is not intended to reduce respiratory disease
due to CHV-1 infection.
Canine Influenza Virus
Inactivated, parenteral vaccines are available for reduction of
disease and shedding caused by H3N8 CIV.56 One vaccine also
reduced the severity of illness caused by co-challenge with CIV
and Streptococcus equi subspecies zooepidemicus.4 The use of
these vaccines could be considered for dogs that are likely to
contact other dogs in regions where CIV is endemic, especially
those that enter boarding or pet daycare facilities. Vaccination
against CIV is required for importation of North American dogs
to Australia.57 The initial vaccine can be given as early as 6
weeks of age. Because CIV vaccines are inactivated, two initial
doses are required 3 to 4 weeks apart, and maximum immunity
does not occur until 1 week after the second dose. As a result,
CIV vaccines may not protect dogs that enter shelters where
canine influenza is endemic, unless newly introduced dogs are
separated from dogs that might be shedding CIV until immuni-
zation is complete. Annual boosters are recommended for dogs
that remain at risk of infection, but the maximum duration of
immunity is unknown.
Canine Parainfluenza Virus
Intranasal vaccination with CPiV significantly reduces clinical
signs and virus shedding after challenge.58-60 One study showed
that intranasal CPiV vaccination reduced clinical signs even
when dogs were challenged as long as 1 year after immuniza-
tion.58 Few studies have compared the efficacy of parenteral
and intranasal vaccines. In a study reported in the early 1980s,
the parenteral vaccine was less effective at reducing shedding
than the intranasal vaccine,59 but a study reported in the 1970s
showed reduction of clinical signs and shedding after parenteral
vaccination.61 The use of one parenteral CPiV vaccine resulted
177
in antibody titers that persisted for at least 2 years.62 Use of
intranasal CPiV vaccines, as opposed to parenteral vaccines, has
been advocated, because they produce local immunity, reduce
shedding, and can be used in puppies as young as 3 to 4 weeks
of age.63 However, proper administration of mucosal vaccines
may be difficult in aggressive dogs or dogs that refuse to be
restrained. Mucosal vaccines may also be associated with tran-
sient respiratory illness for 3 to 10 days after immunization in
a small percentage of dogs. This may be problematic in shelter
and boarding environments, because these signs cannot be dis-
tinguished from those that result from natural infection with
wild-type viruses. More studies that evaluate the relative effi-
cacy of mucosal and parenteral CPiV vaccines are required.
Prevention
Although vaccination can reduce the prevalence of respira-
tory disease in dogs,3 current vaccines do not provide protec-
tion against all the organisms that cause respiratory disease in
dogs, and immunity is not sterile, so infection and mild clini-
cal signs can still occur. When factors such as stress, immuno-
suppression, co-infections, and overwhelming challenge doses
are present, vaccine-induced protection may be overwhelmed.
Prevention of transmissible respiratory tract disease in dogs
therefore involves not only vaccination, but also control meth-
ods that include quarantine of dogs introduced into densely
populated environments, early identification and isolation of
dogs with signs of respiratory disease through proper training
of shelter personnel, avoidance of overcrowding and mixing of
dogs, use of solid walls between runs, reduction of time dogs
spend in a shelter environment, optimum nutrition, fomite con-
trol, control of noise such as barking, and proper ventilation
and disinfection (see Box 11-5). Distances traveled by aerosols
generated by dogs are unknown. Dogs with respiratory disease
should be separated from other dogs in shelter environments
by at least 25 feet (or as far as possible) and ideally placed in
an enclosed room with a separate ventilation system. Isolated
dogs should not be moved back into the general shelter popula-
tion, but adopted out of isolation or a separate recovery room.
Provided proper cleaning and contact times are used, use of dis-
infectants with activity against parvovirus should also kill respi-
ratory viruses, with CAV-2 being the respiratory virus that is
most resistant to disinfection. The order of inspection of dogs
in shelters should be healthy dogs, then quarantined dogs, and
finally dogs in isolation.
When outbreaks occur, attempts should be made to iden-
tify the pathogen(s) involved. This allows potential routes of
transmission of infection and shedding patterns to be identified,
proper vaccination and control strategies to be implemented,
and adequate disinfectants to be selected. For example, quar-
antine for 2 weeks and isolation or removal of any dogs that
develop illness may be effective for diseases such as canine influ-
enza, because of the short incubation and shedding periods.
However, it is less likely to be successful for distemper and bor-
detellosis, which can have prolonged incubation and shedding
periods. For some viral respiratory diseases such as canine influ-
enza, discontinuation of intake and adoptions for 2 to 3 weeks
may be necessary while the disease runs its course. Exposed
dogs could also be transferred to foster care in a household with
no other dogs. Because of a lack of adequate resources for treat-
ment, diagnostic evaluation, and management, some shelters
have elected to euthanize large numbers of affected dogs.
178 SECTION 1  Viral Diseases
Shelters should provide written information on transmis-
sible respiratory disease to clients who adopt shelter animals,
regardless of the presence or absence of clinical signs at the time
of adoption, and communicate fully about diseases that are
endemic in the shelter. Whenever possible, dogs that leave shel-
ters should be kept away from other dogs for at least 2 weeks
(consider 4 weeks if bordetellosis is endemic), and informed
about follow-up medical care. Other dogs in the household
should be vaccinated before they are placed in contact with the
shelter animal. Clients should also be instructed to ensure their
local veterinary clinic is aware that their dog is newly adopted
from a shelter, so that precautions can be taken to prevent con-
tamination of the hospital environment when the dog is exam-
ined a few days after leaving the shelter.
CASE EXAMPLE
Signalment: “Winnie”, a 10-year old male neutered miniature
Schnauzer from Sacramento, CA
History: Winnie was brought to his local veterinarian be-
cause of a 1-day history of serous ocular and nasal dis-
charge, and sneezing. Fever (103.5°F or 39.7°C) was
documented on physical examination, and enrofloxacin
(2 mg/kg PO q24h), bacitracin-neomycin-polymyxin oph-
thalmic ointment (both eyes q8h), and diphenhydramine
(2.2 mg/kg PO q12h) were prescribed, without clinical im-
provement. A day later, he became lethargic, inappetent,
and polydipsic, and the owners described continuous
nasal discharge, and a moist cough. Winnie was brought
to the UC Davis VMTH for a second opinion. He had been
vaccinated regularly for CDV, CAV-2, CPV, rabies, Borde-
tella bronchiseptica, and CPiV. He was recently cared for in
a boarding facility for a week and had been home for the
past 11 days. He also regularly visited dog parks. There
were two other miniature Schnauzers at home, and both
were currently well.
Current Medications: Enrofloxacin, 2 mg/kg PO q24h
Other Medical History: Increased activity of serum ALP was
noted at a senior care visit 2 months before the onset of
respiratory signs.
Physical Examination:
Body Weight: 9.4 kg
General: Lethargic but hydrated. T = 100.8°F (38.2°C),
HR = 104 beats/min, respiratory rate = 12 breaths/min,
mucous membranes pink, CRT = 1 to 2 s.
Eyes, Ears, Nose, and Throat: Severe chemosis, hyperemia,
and mucoid ocular discharge were present bilaterally.
There was also profuse bilateral serous nasal discharge and
decreased nasal airflow, hypersalivation, and right tonsillar
enlargement.
Musculoskeletal: Body condition score 5/9.
Respiratory: Stertorous respiratory noises were noted.
Increased respiratory effort with a normal respiratory rate
was also present. Auscultation revealed referred wheezing
noises in all lung fields.
Gastrointestinal: Tense abdomen, hepatomegaly was
detected on abdominal palpation.
Public Health Aspects
Viruses related to simian virus 5 (parainfluenza virus 5) have
been detected in humans, but the role of CPiV as a human
pathogen is controversial. Reoviruses have a broad host range,
and human infection has been associated with enteritis and
respiratory disease; MRV-3 infection was associated with men-
ingitis in a child.64 Thus they represent a potential zoonosis.
Although adenoviruses, influenza viruses, coronaviruses, and
paramyxoviruses cause respiratory disease in humans, there is
no evidence that CAV-2, CIV, CDV, CHV-1, or CRCoV can be
transmitted to humans. Human influenza viruses such as H1N1
can cause disease in dogs and may have the potential to spread
from infected dogs back to humans.
All Other Systems: No abnormalities were detected.
Ophthalmologic Examination: Pupillary light reflexes
(PLRs) (direct and consensual) were brisk and complete. There
was no evidence of anisocoria. Menace response, dazzle,
and palpebral reflexes were all complete. Globe position
and movements were normal bilaterally (OU). Periorbital
palpation and globe retropulsion were also normal. The dog
behaved as if sighted. The eyelids were normal. Both nictitans
were hyperemic and edematous, and there was moderate
conjunctival hyperemia, chemosis, and mucoid ocular
discharge OU. Bilateral iris atrophy and lens nuclear sclerosis
were also present. The cornea, anterior chamber, vitreous,
and dilated fundic examination were normal OU. In the left
eye (OS), a pinpoint incipient anterior cortical cataract was
identified just ventral to the center of the lens. In the right
eye (OD), a linear pigment deposition on the anterior lens
capsule was present at the 5 o’clock position.
Schirmer Tear Test: 8 mm/min OU
Intraocular Pressure: 16 mm Hg OS, 17 mm Hg OD
Fluorescein Stain: Multiple dendritic ulcers were identified at
the ventral peripheral cornea OD (see Figure 17-2).
Laboratory Findings:
CBC:
HCT 53.5% (40-55%)
MCV 67.7 fL (65-75 fL)
MCHC 35.3 g/dL (33-36 g/dL)
1 nucleated red cell/100 WBCs
WBC 14,600 cells/µL (6000-13,000 cells/µL)
Neutrophils 10,804 cells/µL (3000-10,500 cells/µL)
Band neutrophils 438 cells/µL
Lymphocytes 1752 cells/µL (1000-4000 cells/µL)
Monocytes 1460 cells/µL (150-1200 cells/µL)
Eosinophils 0 cells/µL (0-1500 cells/µL)
Platelets 216,000 platelets/µL (150,000-400,000 platelets/µL)
A few highly reactive lymphocytes were noted.
Serum Chemistry Profile:
Sodium 142 mmol/L (145-154 mmol/L)
Potassium 6.1 mmol/L (3.6-5.3 mmol/L)
Chloride 102 mmol/L (105-116 mmol/L)
Bicarbonate 25 mmol/L (16-26 mmol/L)
Phosphorus 6.7 mg/dL (3.0-6.2 mg/dL)
Calcium 9.5 mg/dL (9.9-11.4 mg/dl)
BUN 20 mg/dL (8-31 mg/dL)
Creatinine 0.7 mg/dL (0.5-1.6 mg/dL)
 CHAPTER 17  Canine Viral Respiratory Infections
Glucose 80 mg/dL (60-104 mg/dL)
Total protein 5.7 g/dL (5.4-7.4 g/dL)
Albumin 2.2 g/dL (2.9-4.2 g/dL)
Globulin 3.5 g/dL (2.3-4.4 g/dL)
ALT 564 U/L (19-67 U/L)
AST 116 U/L (21-54 U/L)
ALP 1024 U/L (15-127 U/L)
Gamma GT 10 U/L (0-6 U/L)
Cholesterol 195 mg/dL (135-345 mg/dL)
Total bilirubin 0.4 mg/dL (0-0.4 mg/dL).
Urinalysis: SGr 1.009; pH 8.5, negative for protein, bilirubin,
hemoprotein, glucose; rare WBC/HPF, 0 RBC/HPF, 0-3 hyaline
casts/HPF, few amorphous crystals, moderate degenerated
cells and amorphous debris in the sediment.
Imaging Findings:
Thoracic and Cervical Radiographs: A mild to moderate
bronchial and interstitial pattern was identified and
was most prominent in the caudal lung fields. Cervical
radiographs were unremarkable. The pulmonary changes
were considered nonspecific, but more significant than
those expected due to age.
Abdominal Ultrasound: The cranial pole of the right adrenal
was mildly to moderately enlarged. The liver appeared
sonographically normal. A 3 mm stone was identified in the
urinary bladder.
Microbiologic Testing: A nasal swab specimen was
submitted for real-time PCR panel for canine respiratory
pathogens, which included CHV-1, CIV, CDV, CPiV, CAV-2,
and B. bronchiseptica. The results were available 3 days later,
and the specimen was positive for CHV-1 DNA.
Diagnosis: Upper respiratory disease and keratitis associated
with CHV-1 infection.
Treatment: Winnie was hospitalized in isolation and treated
with 0.9% NaCl (30 mL/hr IV), nebulization for 15 min
q8h, clavulanic acid-amoxicillin (13.5 mg/kg PO q12h),
and lubricating eye ointment (1 inch OU q4h). Clinical
improvement occurred a day later, electrolyte abnormalities
normalized, and the dog was discharged with instructions
SUGGESTED READINGS
Buonavoglia C, Martella V. Canine respiratory viruses. Vet Res.
2007;38:355-373.
Crawford PC, Dubovi EJ, Castleman WL, et al. Transmission of equine
influenza virus to dogs. Science. 2005;310:482-485.
Ellis JA, Krakowka GS. A review of canine parainfluenza virus infection
in dogs. J Am Vet Med Assoc. 2012;240:273-284.
Erles K, Dubovi EJ, Brooks HW, et  al. Longitudinal study of viruses
associated with canine infectious respiratory disease. J Clin Micro-
biol. 2004;42:4524-4529.
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179
to administer topical idoxuridine (0.1% ophthalmic solution,
2 drops OU q4-6h). Unfortunately a caretaker of the dog
did not regularly administer the idoxuridine. At a recheck 5
weeks after the dog was first seen, the owner reported 80%
improvement in Winnie’s respiratory signs, but he continued
to sneeze approximately six times a day and was pawing at
his eyes. Examination revealed persistent conjunctivitis, and
a fluorescein stain showed large, superficial areas of uptake
over the central cornea OU (see Figure 17-2). A Schirmer
tear test (STT) was 13 mm/min OS and 8 mm/min OD. A CBC
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persistently increased ALP (835 U/L) and GGT activities
(12 U/L) and improvement in the serum ALT activity (124
U/L). The owner was instructed to administer idoxuridine
for an additional 2 weeks, as well as lubricating ointment,
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lysine gel (2 mL [400 mg] q12h PO), separating the ophthalmic
medications with administration of the idoxuridine first. At a
recheck 2 weeks later, all signs had resolved and there was
no fluorescein uptake. STT results were 14 mm/min OS and
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had another episode of respiratory signs 4 months later, at
which time the STT results were 15 mm/min OU. Treatment
with lubricating eye ointment and l-lysine was continued.
Comments: This was an unusual case of ocular and respiratory
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results for other respiratory pathogens did not rule out
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organisms may have ceased or been at undetectable levels.
Although idoxuridine was used to treat this dog, topical
cidofovir may also have been effective.50 Approximately 2
years later, mild serous nasal discharge and depigmentation
of the planum nasale developed. A biopsy of the nasal
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180 SECTION 1  Viral Diseases
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 CHAPTER 17  Canine Viral Respiratory Infections 181

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CHAPTER 18
Infectious Canine Hepatitis
Jane E. Sykes
Overview of Infectious Canine Hepatitis
First Described: First described as infectious canine hepatitis
(ICH) in 1947 in Sweden by Rubarth1; CAV-1 was first iso-
lated in chick embryos in 19512
Cause: Canine adenovirus-1 (family Adenoviridae, genus
Mastadenovirus)
Affected Hosts: Dogs, coyotes, foxes, wolves, bears, skunks
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact with infected saliva,
feces and urine, and contaminated fomites
Major Clinical Signs in Dogs: Fever, lethargy, inappetence, vom-
iting, hemorrhagic diarrhea, abdominal pain, dehydration,
conjunctivitis, petechial hemorrhages, tachypnea, cough,
corneal edema (“blue eye”), rarely icterus or neurologic signs.
Differential Diagnoses: Other systemic viral diseases such
as parvoviral enteritis and canine distemper, enteric viral
and bacterial infections, hepatotoxicosis (e.g., mush-
rooms), Rocky Mountain spotted fever, gastrointestinal
foreign body, dietary indiscretion, leptospirosis, porto-
systemic shunting with hepatic encephalopathy, dissemi-
nated fungal infections (especially systemic candidiasis),
systemic protozoal infections (especially sarcocystosis,
toxoplasmosis or African trypanosomiasis), hemic neopla-
sia (especially lymphoma).
Human Health Significance: CAV-1 does not infect humans.
Etiology and Epidemiology
Infectious canine hepatitis (ICH) is an uncommonly recog-
nized disease of dogs that is caused by canine adenovirus type 1
(CAV-1), a non-enveloped, icosahedral double-stranded DNA
virus that is antigenically related to CAV-2 (see Chapter 17) (Fig-
ure 18-1). CAV-1 also causes disease in wolves, coyotes, skunks,
and bears, as well as encephalitis in foxes, but the diversity of
wildlife hosts is not as great as that for CDV. Ferrets are not
susceptible.3 ICH has also been referred to as Rubarth’s disease,
after Carl Sven Rubarth, a veterinarian who first described the
disease in the late 1940s.1 Over the subsequent 10 years, ICH was
described worldwide, including the United States, Canada, United
Kingdom, Australia, Japan, Brazil, and throughout Europe. The
virus can survive for months at room temperature, but should be
readily inactivated by disinfectants with activity against canine
parvovirus (CPV).4 Disease most commonly occurs in dogs that
are less than 1 year of age, but was reported in adult dogs before
widespread vaccination for ICH was introduced.
182
The strong antigenic relationship between CAV-1 and CAV-2 is
clinically important, because vaccines that contain CAV-2 protect
against infection with CAV-1 and vice versa. After the introduc-
tion of CAV-1 vaccines, ICH largely disappeared, but over the past
decade it has reemerged, with published reports of disease from
Italy, Switzerland, and the United States.5-8 Disease in Europe has
been associated with puppy trading from kennels in eastern Euro-
pean countries and possibly spillover of virus circulating in wild-
life.9 In Italy, three outbreaks occurred in shelters in southern Italy,
and the others involved purebred puppies imported from Hungary
a few days before the onset of clinical signs. Several of the dogs
were co-infected with other viruses, such as canine distemper virus
(CDV), CPV, or canine enteric coronavirus. Encephalopathy due
to CAV-1 infection was described in nine 5-week-old Labrador
retriever puppies from Arkansas in the United States, all of which
belonged to the same litter.8 The litter was from an unvaccinated
bitch. These case descriptions confirm that CAV-1 continues to
circulate in the dog population and can result in severe disease in
young dogs when vaccination does not occur, is improperly timed,
and stress, co-infections, and overcrowded conditions prevail.
Clinical Features
Signs and Their Pathogenesis
CAV-1 is shed in saliva, feces, and urine, and transmission occurs
through direct dog-to-dog contact or contact with contaminated
fomites such as hands, utensils, and clothing. Ectoparasites such
as fleas and ticks are also potential mechanical vectors.4 Airborne
transmission does not appear to be important. Initial infection
occurs through the nasopharyngeal, conjunctival, or oropha-
ryngeal route, and the virus replicates within the tonsils, after
which it spreads to regional lymph nodes and the bloodstream
via lymphatics. Subsequently, infection of hepatocytes and
endothelial cells within a variety of tissues occurs, such as the
lungs, liver, kidneys, spleen, and eye with resultant hemorrhage,
necrosis, and inflammation. The virus replicates in the nucleus
of host cells, where crystalline arrays of virions form. There is
severe condensation and margination of nuclear chromatin, with
inclusion body formation (Figure 18-2). The virions are released
by cell lysis, which leads to tissue injury and disseminated intra-
vascular coagulation (DIC). Within the liver, the virus initially
infects Kupffer’s cells and subsequently spreads to hepatocytes.
Clinical signs generally occur after an incubation period of
4 to 9 days, although many dogs probably show no signs of ill-
ness.4 Three overlapping disease syndromes have been described.
The first is peracute disease with circulatory collapse, coma, and
death after a brief illness that lasts less than 24 to 48 hours.
The second, most commonly described syndrome is acute dis-
ease, which is associated with high morbidity and reported mor-
tality rates of around 10% to 30%.4 Dogs with acute disease

FIGURE 18-1  Structure of canine adenovirus. The virus is a non-enveloped, lcosahedral
virus with fibers (purple) that radiate outwards from the virion.
FIGURE 18-2  Infection of hepatocytes and endothelial cells with CAV-1 produces
characteristic basophilic intranuclear inclusions surrounded by a clear zone that separates
them from the marginated chromatin (arrow). H&E stain. (Courtesy Dr. W. Crowell, College
of Veterinary Medicine, The University of Georgia and Noah’s Archive, College of Veterinary
Medicine, The University of Georgia. In Zachary JF, McGavin M. Pathologic Basis of Veteri-
nary Disease, 5 ed. St. Louis, MO: Mosby; 2012.)
either recover or die within a 2-week period. The third is a more
chronic form that occurs in dogs with partial immunity, with
death due to hepatic failure weeks (subacute disease) or months
(chronic infection) after initial infection.10
Acute disease is variably characterized by the presence of
fever, tonsillitis, conjunctivitis, inappetence, lethargy, weakness,
polydipsia, vomiting, hematemesis, diarrhea, cough, tachypnea,
and icterus. Diarrhea may contain frank blood or melena. Wide-
spread petechial and ecchymotic hemorrhages and hematuria
can be seen. Corneal edema (“blue eye”) occurs in the first week
of illness and results from replication of virus within corneal
endothelial cells (Figure 18-3). Rarely, neurologic signs such
CHAPTER 18  Infectious Canine Hepatitis 183
FIGURE 18-3  Young adult dog with corneal edema from an Italian shelter outbreak
of CAV-1 infection. (Courtesy Dr. Nicola Decaro, Department of Veterinary Public Health,
Faculty of Veterinary Medicine of Bari, Italy.)
as seizures, ataxia, circling, apparent blindness, head pressing,
and nystagmus have been reported in association with CAV-1
encephalitis.7,8 The development of neurologic signs may also
represent hepatic encephalopathy, intracranial thrombosis or
hemorrhage, or, as occurred in one outbreak, concurrent infec-
tion with CDV.7
The antibody response appears 7 days after infection and
limits tissue damage. Viral persistence within the renal glom-
eruli, uveal structures of the eye (the iris and ciliary body), and
the cornea can trigger immune complex formation in dogs that
recover from acute illness. This leads to glomerulonephritis with
proteinuria, severe uveitis, and persistent corneal edema in some
surviving dogs. Glomerulonephritis usually occurs about 1 to
2 weeks after the acute signs resolve. Glomerular lesions contain
deposits of viral antigen, IgG, IgM, and C3.11,12 Infection of
the glomerular endothelium is followed by a persistent tubular
infection, development of interstitial nephritis, and viruria, but
chronic renal failure has not been described. Viral shedding in
the urine can occur for up to 6 to 9 months after infection. Ante-
rior uveitis is associated with massive influx of inflammatory
cells into the anterior chamber. Occasionally persistent corneal
edema fails to resolve for months and may be associated with
complications such as glaucoma.13 The Afghan hound is report-
edly susceptible to this complication.14
In experimental infections with CAV-1, chronic hepatitis
with extensive fibrosis was observed in some dogs that recovered
from acute illness, with survival for up to 8 months.10 The virus
could not be found in hepatic lesions from these dogs. Attempts
to detect CAV-1 in the liver of other dogs with chronic active
hepatitis using PCR assays have to date been unrewarding.15-17
Physical Examination Findings
Physical examination findings in dogs with acute ICH vary, but
might include lethargy, dehydration, fever (up to 106°F or 41°C),
184 SECTION 1  Viral Diseases
TABLE 18-1
Diagnostic Assays Available for Infectious Canine Hepatitis
Assay Specimen Type Target
Histopathology Usually necropsy Hepatic necrosis with in- True sensitivity and specificity of inclusion visualization
­specimens, but also tranuclear inclusions;
liver biopsy CAV-1 antigen with
immunohistochemistry
Polymerase chain Blood; rectal, CAV-1 DNA
reaction (PCR) ­conjunctival and
nasal swabs; urine;
tissues collected at
necropsy
Virus isolation All body secretions, CAV-1
tissues
CAV, Canine adenovirus.
congestion and enlargement of the tonsils (which may be severe),
pallor, conjunctivitis, peripheral lymphadenopathy, tachypnea,
increased lung sounds, and tachycardia. In some reports, serous
to mucopurulent ocular and nasal discharge have been observed,
but these may have resulted from co-infections with other respi-
ratory viruses.4 Abdominal palpation may reveal hepatomegaly,
splenomegaly, or abdominal pain. Icterus is uncommon but can
occur in dogs with a more prolonged course of disease. Peripheral
edema that involves the head, neck, and ventral abdomen has
been described.18 Puppies with CAV-1 encephalitis may show
signs of circling, vocalization, head pressing, ataxia, and blind-
ness.8 Coagulopathies may be manifested as cutaneous or muco-
sal petechial hemorrhages; gingival hemorrhages; epistaxis; or
prolonged bleeding from venipuncture sites.
Ocular complications occur in at least 20% of affected dogs.
Unilateral or, less commonly, bilateral corneal edema may be
observed, which initially develops at the limbus and is occasion-
ally associated with blepharospasm, photophobia, and a serous
ocular discharge.13 Unilateral involvement may progress to
bilateral involvement over several days. Using a slit lamp, the
cornea is markedly thickened, and there is episcleral and ciliary
injection. Uveitis may also be apparent. Corneal ulceration and
increased intraocular pressure has also been described in dogs
with corneal edema, the latter of which may result in blindness.13
Diagnosis
ICH should be suspected in any dog less than 1 year of age that
has a questionable vaccination history and signs of fever, respi-
ratory, gastrointestinal, and hepatic disease, and certainly in
any young dog that develops corneal edema. Diagnosis is easily
achieved at necropsy when characteristic intranuclear inclusion
bodies are seen in tissues, but the sensitivity and specificity of this
finding is unknown. Inclusion bodies may be seen on impression
smears of liver biopsies or tissue obtained at necropsy. At the
time of writing, antemortem diagnosis is challenging owing to
the lack of commercially available assays that specifically detect
the virus or viral DNA, and the disease may be underdiagnosed
(Table 18-1). Serologic assays are available, but they provide
Performance
unknown. Liver biopsy may not be feasible because of
coagulopathies.
Sensitivity and specificity may vary depending on assay
design. Specific CAV-1 assays are not widely offered by
commercial veterinary diagnostic laboratories. Some
assays differentiate between vaccine (CAV-2) and field
virus (CAV-1). The significance of a positive result from
urine may be difficult to interpret due to subclinical
shedding. False-negative results may occur as a result of
PCR assay inhibition by components of feces or in dogs
with subacute or chronic presentations.
Sensitive and specific, but generally only available as a
research tool.
only retrospective diagnosis, and interpretation of these assays
may be a challenge when there is a history of vaccination.
Laboratory Abnormalities
Complete Blood Count
Findings reported on the CBC are variable and include leuko-
penia, anemia, increased nucleated red blood cells, and mod-
erate to severe thrombocytopenia. Leukopenia occurs early in
the course of infection and may be profound. Initially there is
a lymphopenia, after which neutropenia occurs and worsens
progressively until death.18 Increased band neutrophils and
toxic changes may also be present. Occasionally leukocytosis is
observed.19 Leukocytosis and lymphocytosis may occur as part
of the recovery process.
Serum Biochemical Tests
Changes on the serum biochemistry profile include increased
activity of serum ALT (sometimes >1000 U/L) and ALP, hyper-
bilirubinemia, hypoglycemia, and hypoalbuminemia.20 Determi-
nation of serum ammonia concentration may facilitate diagnosis
of hepatic encephalopathy in dogs with neurologic signs.
Urinalysis
The urinalysis of dogs with ICH may reveal proteinuria, hyaline
and granular cylindruria, hematuria, and bilirubinuria.
Coagulation Profile
In addition to thrombocytopenia, coagulation abnormalities
reported in ICH reflect the presence of DIC and hepatic failure
and include prolonged prothrombin time, markedly prolonged
activated partial thromboplastin time (to 6 to 7 times control
values), decreased factor VIII activity, hypofibrinogenemia, and
increased fibrinogen degradation products. Platelet function
assays have shown reduced platelet adhesion.18
Diagnostic Imaging
Findings on plain radiography and abdominal sonography in
dogs with ICH have not been reported. Plain radiography might
reveal a normal to slightly enlarged liver, and poor detail as a

result of abdominal effusion or the young age (and therefore
low intra-abdominal fat content) of affected dogs.
Microbiologic Tests
Virus Isolation
CAV-1 can be readily isolated in a variety of cell types, such
as Madin-Darby canine kidney cells.7,8 In dogs with acute ill-
ness, any body fluid or tissue is likely to contain sufficient virus
for isolation. Cultures are evaluated for a cytopathic effect with
intranuclear inclusions, and the presence of CAV-1 is confirmed
using immunostaining. Isolation is not widely offered by com-
mercial veterinary diagnostic laboratories.
Serologic Diagnosis
Serologic tests are available commercially for detection of IgG
and IgM against CAV-1, which include ELISA assays, hem­
agglutination-inhibition, and serum neutralization. Unfortu-
nately, dogs with acute disease may die before they develop
antibodies to the virus.10 For dogs that recover from illness,
a recent history of vaccination may complicate interpretation
of acute and convalescent phase serology. In the absence of a
vaccination history, a fourfold rise in titer over a 2- to 3-week
period together with compatible clinical signs is supportive of
the diagnosis of ICH. Titers that follow natural infection may
be higher than those that follow vaccination.
Molecular Diagnosis Using the Polymerase Chain Reaction
Conventional PCR assays for detection of CAV-1 in clinical speci-
mens such as nasal, rectal, and ocular swabs and blood, as well as
tissue obtained at necropsy, have been described. These include
assays that differentiate between CAV-1 and CAV-221 and repre-
sent one of the most rapid means of antemortem diagnosis. Because
of the rarity of the disease, the clinical sensitivity and specificity
of these assays are not well understood. Real-time PCR assays
that specifically detect CAV-1 infection were not available at the
time of writing (see Chapter 5 for a discussion of the differences
between conventional and real-time PCR assays). The results of
PCR assays on urine may be more difficult to interpret than those
for other specimens, because of the potential for chronic shedding
from this site in the absence of clinical signs. Assays that specifi-
cally detect CAV-1 differentiate between virulent CAV-1 virus and
vaccine virus, because vaccines for ICH all contain only CAV-2.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with ICH include blood-
tinged ascites or hemoabdomen; a slightly enlarged, congested
or mottled liver; mild splenomegaly; enlarged, congested, and
edematous lymph nodes; and fibrin deposition on the surface of
abdominal viscera (Figure 18-4). The gallbladder wall is typi-
cally markedly thickened and edematous. Petechial and ecchy-
motic subserosal hemorrhages may be apparent in multiple
viscera, and the intestinal tract may contain bloody fluid. The
brain may also contain petechial hemorrhages or areas of gray
discoloration.8 Parenchymal organs may contain fibrin thrombi.
Occasionally, multifocal pulmonary consolidation and/or sero-
sanguineous pleural fluid is noted.
Histopathologic Findings
Histopathologic findings are variable and depend on the course
of infection and possibly the virus strain. The most characteris-
tic finding is hepatocellular necrosis and intranuclear viral inclu-
sion bodies within Kupffer’s cells and hepatocytes, and a mixed
CHAPTER 18  Infectious Canine Hepatitis 185
FIGURE 18-4  Infectious canine hepatitis, hepatic necrosis, liver, dog. The liver from
dogs with ICH can be slightly enlarged and friable with a blotchy yellow discoloration.
Sometimes fibrin is evident on the capsular surface. Note the petechiae on the serosal sur-
face of the intestines caused by vascular damage. (Courtesy Dr. M.D. McGavin, College of
Veterinary Medicine, University of Tennessee. In Zachary JF, McGavin MD. Pathologic Basis
of Veterinary Disease, 5 ed. St. Louis, MO: Mosby; 2012.)
inflammatory cell infiltrate (see Figure 18-2). Fibrosis may be
observed in dogs with chronic liver injury. Interstitial nephri-
tis, with focal accumulations of neutrophils, mononuclear cells,
and fibrosis, may also be present,22 as well as evidence of wide-
spread hemorrhage, thrombosis, and necrosis as a result of DIC.
Findings in dogs with CAV-1 encephalitis have included mild
spongiosis, neuronal necrosis, hemorrhage, and perivascular
cuffing with mononuclear cells.8 Viral inclusion bodies may be
found in endothelial cells of meningeal vessels, the cornea, renal
glomeruli, and the tonsils. Immunohistochemistry can be used
to confirm the presence of the virus within tissues.
Treatment and Prognosis
Supportive Care
Treatment of dogs with acute ICH is purely supportive and con-
sists primarily of fluid therapy, including crystalloid fluids and
blood products. Fluid therapy should be aggressive with careful
monitoring and avoidance of overhydration, because of increased
vascular permeability and hypoalbuminemia. Fluids should be
supplemented with electrolytes and dextrose as required. Other
medications that may be indicated include antiemetics, antacids,
sucralfate, whole blood or plasma transfusions, and colloids such
as hetastarch. Partial or total parenteral nutrition may be indicated
for severely affected dogs that do not tolerate enteral feeding.
Dogs with DIC may require treatment with heparin in addition
to plasma. Management of hepatic encephalopathy with lactulose
enemas, oral lactulose (in the absence of vomiting), and poorly
absorbed oral antimicrobial drugs such as ampicillin may also be
indicated. The use of parenteral broad-spectrum antimicrobial
drugs should be considered for dogs with hemorrhagic gastroen-
teritis that may develop bacteremia as a result of bacterial translo-
cation. After fluorescein staining has shown no evidence of corneal
ulceration, dogs with severe corneal edema and uveitis should be
treated with topical ophthalmic preparations that contain gluco-
corticoids and atropine to prevent development of glaucoma.
186 SECTION 1  Viral Diseases
Prognosis depends on the severity of disease, which reflects
the immune status of the affected dog and possibly the virus
strain. There is a possibility that recovered dogs may develop
chronic hepatitis or chronic glomerulonephritis, but the extent
to which this truly occurs is unknown.
Immunity and Vaccination
Immunity to natural infection with CAV-1 is probably lifelong.
Effective vaccines have been available and widely used as part
of core vaccine programs for dogs for many years. Immunity
after immunization with attenuated live vaccines lasts at least
3 years and probably longer. Early vaccines, which contained
CAV-1, were associated with the development of corneal edema
and glomerulonephritis in a small percentage (<1%) of immu-
nized dogs. Replacement of attenuated live CAV-1 with CAV-2
occurred after 1980 and eliminated this complication.23 These
vaccines have the potential to cause transient respiratory signs
and tonsillitis in dogs if accidental inhalation occurs, so care
should be taken not to aerosolize the vaccine during administra-
tion. Maternal antibody persists until puppies are 12 weeks of
age and interferes with immunization when virus neutralization
titers exceed 1:100. Vaccines should be administered every 3 to
4 weeks from 6 weeks of age, with the last vaccine given no ear-
lier than 16 weeks of age (see Appendix). Vaccine virus may be
shed from the respiratory tract by dogs after vaccination, which
has the potential to immunize other dogs as well. It has been
suggested that this phenomenon may have been responsible for
the virtual disappearance of the disease in the dog population in
regions where vaccination is widely performed.24
Prevention
The best means of prevention of ICH is proper vaccination.
Additional control measures that could be considered in loca-
tions where outbreaks occur, such as in shelters, include proper
disinfection, isolation, and prevention of overcrowding and
other co-infections, which may worsen disease. Because contact
with wild animal species such as coyotes, wolves, and foxes that
might be shedding the virus may also be a source of infection for
dogs, exclusion of these species from interactions with domestic
dogs may also serve to prevent the disease.
Public Health Aspects
There is no evidence that CAV-1 infects humans.
SUGGESTED READINGS
Decaro N, Campolo M, Elia G, et  al. Infectious canine hepatitis: an
“old” disease reemerging in Italy. Res Vet Sci. 2007;83:269-273.
Gocke DJ, Preisig R, Morris TQ, et al. Experimental viral hepatitis in
the dog: production of persistent disease in partially immune animals.
J Clin Invest. 1967;46:1506-1517.
REFERENCES
1. Rubarth S. An acute virus disease with liver lesion in dogs (hepatitis
contagiosa canis): a pathologico-anatomical and etiological investi-
gation. Acta Path Microbiol Scand. 1947(Suppl):69.
2. Miles JA, Parry HB, Larin NM, et al. Cultivation of canine hepati-
tis virus in embryonated hen’s eggs and its subsequent transmission
to dogs. Nature. 1951;168(4277):699-700.
3. Hudson JR, Mansi W. Rubarth’s disease (canine virus hepatitis)
II. The insusceptibility of ferrets to experimental infection. J Comp
Pathol. 1953;63:335-345.
4. Cabasso VJ. Infectious canine hepatitis virus. Ann N Y Acad Sci.
1962;101:498-514.
5. Pratelli A, Martella V, Elia G, et  al. Severe enteric disease in an
animal shelter associated with dual infections by canine adenovirus
type 1 and canine coronavirus. J Vet Med B Infect Dis Vet Public
Health. 2001;48:385-392.
6. Muller C, Sieber-Ruckstuhl N, Decaro N, et al. [Infectious canine
hepatitis in 4 dogs in Switzerland]. Schweiz Arch Tierheilkd.
2010;152:63-68.
7. Decaro N, Campolo M, Elia G, et al. Infectious canine hepatitis: an
“old” disease reemerging in Italy. Res Vet Sci. 2007;83:269-273.
8. Caudell D, Confer AW, Fulton RW, et al. Diagnosis of infectious
canine hepatitis virus (CAV-1) infection in puppies with encepha-
lopathy. J Vet Diagn Invest. 2005;17:58-61.
9. Decaro N. Infectious canine hepatitis—a re-emerging disease.
Sevilla, Spain: 21st ECVIM-CA Congress; 2011:78–79.
10. Gocke DJ, Preisig R, Morris TQ, et al. Experimental viral hepatitis
in the dog: production of persistent disease in partially immune
animals. J Clin Invest. 1967;46:1506-1517.
11. Morrison WI, Nash AS, Wright NG. Glomerular deposition of
immune complexes in dogs following natural infection with canine
adenovirus. Vet Rec. 1975;96:522-524.
12. Hervas J, Gomez-Villamandos JC, Perez J, et al. Focal mesangial-
sclerosing glomerulonephritis and acute-spontaneous infectious
canine hepatitis: structural, immunohistochemical and subcellular
studies. Vet Immunol Immunopathol. 1997;57:25-32.
13. Curtis R, Barnett KC. The ocular lesions of infectious canine hepa-
titis. 1. Clinical features. J Small Anim Pract. 1973;14:375-389.
14. Curtis R, Barnett KC. Canine adenovirus-induced ocular lesions in
the Afghan hound. Cornell Vet. 1981;71:85-95.
15. Chouinard L, Martineau D, Forget C, et  al. Use of polymerase
chain reaction and immunohistochemistry for detection of canine
­adenovirus type 1 in formalin-fixed, paraffin-embedded liver
of dogs with chronic hepatitis or cirrhosis. J Vet Diagn Invest.
1998;10:320-325.
16. Boomkens SY, Slump E, Egberink HF, et  al. PCR screening for
candidate etiological agents of canine hepatitis. Vet Microbiol.
2005;108:49-55.
17. Bexfield NH, Andres-Abdo C, Scase TJ, et  al. Chronic hepatitis
in the English springer spaniel: clinical presentation, histological
description and outcome. Vet Rec. 2011;169(16):415.
18. Wigton DH, Kociba GJ, Hoover EA. Infectious canine hepatitis:
animal model for viral-induced disseminated intravascular coagula-
tion. Blood. 1976;47:287-296.
19. Kobayashi Y, Ochiai K, Itakura C. Dual infection with canine dis-
temper virus and infectious canine hepatitis virus (canine adenovirus
type 1) in a dog. J Vet Med Sci. 1993;55:699-701.
20. Beckett SD, Burns MJ, Clark CH. A study of the blood glucose,
serum transaminase, and electrophoretic patterns of dogs with
infectious canine hepatitis. Am J Vet Res. 1964;25:1186-1190.
21. Hu RL, Huang G, Qiu W, et al. Detection and differentiation of
CAV-1 and CAV-2 by polymerase chain reaction. Vet Res Com-
mun. 2001;25:77-84.
22. Wright NG. Interstitial nephritis in a dog associated with infectious
canine hepatitis virus. Vet Rec. 1970;86:92-93.
23. Bass EP, Gill MA, Beckenhauer WH. Evaluation of a canine adeno-
virus type 2 strain as a replacement for infectious canine hepatitis
vaccine. J Am Vet Med Assoc. 1980;177:234-242.
24. MacLachlan NJ, Dubovi EJ. Adenoviridae. Fenner’s Veterinary
Virology. 4th ed. Elsevier; 2011:203-212.
CHAPTER 19
Feline Panleukopenia Virus Infection
and Other Viral Enteritides
Jane E. Sykes
Overview of Feline Parvoviral Enteritis
First Described: 1928, Verge and Christoforoni, France1
Cause: Feline panleukopenia virus (FPV); also canine parvo-
virus (CPV)-2a, CPV-2b, and CPV-2c (Family Parvoviridae,
subfamily Parvovirinae, genus Parvovirus)
Affected Hosts: Domestic and wild cats; foxes, mink, and
raccoons
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact with virus in feces and
vomitus and contaminated fomites.
Major Clinical Signs: Fever, lethargy, inappetence, vomiting,
diarrhea, dehydration, sudden death. Neurologic signs
(primarily cerebellar signs) may also occur.
Differential Diagnoses: Other feline viral enteritides, toxins,
gastrointestinal foreign body, enteric parasitic infections
such as giardiasis and nematode infections, enteric bac-
terial infections such as salmonellosis, pancreatitis, or
inflammatory bowel disease. Congenital central nervous
system defects should be considered in cats with neuro-
logic signs.
Human Health Significance: FPV is not known to infect
humans, but was isolated from a monkey.
Etiology and Epidemiology
Feline panleukopenia virus (FPV) is a parvovirus that causes
enteritis and panleukopenia in domestic and wild cat species
worldwide.2 It has also been associated with disease in rac-
coons, mink, foxes, and a monkey, and can replicate in ferrets
without causing disease. Feline panleukopenia is sometimes
confusingly referred to as “cat plague” and “feline distemper.”
FPV is a small, single-stranded non-enveloped DNA virus that
is closely related to CPV-2, but in contrast to CPV-2, which
emerged in the late 1970s, the existence of FPV has been known
since the 1920s.1 FPV has the same ability as CPV-2 to survive
long periods in the environment and resist disinfection, and has
the same preference for replication in rapidly dividing cells (see
Chapter 14).
Feline panleukopenia is most likely to occur in cats younger
than 1 year of age, but it can occur in unvaccinated or improp-
erly vaccinated cats of all ages. The median age of affected cats
in one study was 4 months, and when disease occurred in vac-
cinated cats, it occurred only in cats that had not received a
booster vaccine after 12 weeks of age.3 However, kitten deaths
have been reported in households of fully vaccinated kittens,
possibly because of exposure to large amounts of virus in the
environment.4 Outbreaks of panleukopenia in cats correlate
seasonally with increases in susceptible newborn kitten num-
bers. Panleukopenia occurs most commonly in multicat house-
holds, and especially in enclosed, shelter environments. It can
also occur in cats with outdoor exposure, such as barn, feral,
and stray cats. In one study, the prevalence of protective anti-
body titers to FPV in feral cats in Florida was only 33%, which
suggested a low rate of exposure to the virus.5 In some North
American shelters, devastating outbreaks of panleukopenia have
led to euthanasia of large numbers of cats. Contact with other
cats may not be present in the history, because fomite transmis-
sion is so effective.3 FPV replicates to a limited extent in dogs,
without disease or virus shedding. Some feline panleukopenia
results from infection of cats by the related mink enteritis virus,
CPV-2a, CPV-2b, or CPV-2c.6-8 Mixed infections with FPV and
CPV-2 variants have been detected in cats, and there is evidence
for recombination between FPV and CPV-2 variants.7,9 Infec-
tion of cats with CPV-2 variants is uncommon in Europe but
predominated among cats with panleukopenia in Asia.10
Other viral pathogens that have been associated with gas-
troenteritis in cats include feline enteric coronavirus (see
Chapter 20), FeLV, rotaviruses, caliciviruses, reoviruses, and
astroviruses. A torovirus-like agent has been associated with
a ­syndrome of diarrhea and protrusion of the nictating mem-
branes in cats.11 Togavirus-like and picornavirus-like particles
have been identified in the feces of Australian cats, but their
significance is uncertain.12
Clinical Features
Signs and Their Pathogenesis
The pathogenesis of FPV infection is similar to that of CPV
infection (see Chapter 14). Transmission is by the fecal-oral
route, and indirect transmission through contaminated fomites
represents the most important means of infection. Like CPV,
FPV enters cells using transferrin receptors13 and replicates in
cells that are in the S-phase of the mitotic cycle. Initially, the
virus replicates in oropharyngeal lymphoid tissue, after which
it disseminates in blood to all tissues. Infection of lymphoid tis-
sues leads to lymphoid tissue necrosis. Infection of the marrow
is associated with leukopenia, which is compounded by neutro-
phil sequestration in damaged gastrointestinal tissue. The virus
replicates in the intestinal crypt epithelial cells, with shortening
of villi, increased intestinal permeability, and malabsorption.
Subclinical infection is probably widespread, especially in
young adult or adult, immune-competent cats. Disease severity
depends on factors such as age, immune status, and concurrent
187
188 SECTION 1  Viral Diseases

infections with other bacterial or viral pathogens, which increase Kittens with cerebellar signs are generally bright and alert
the turnover rate of intestinal epithelial cells and enhance viral but exhibit intention tremors, incoordination, ataxia, hyper-
replication and cellular destruction. Co-infections can occur with metria, a broad-based stance, decreased postural reactions, a
feline enteric coronavirus, Clostridium piliforme, Salmonella truncal sway, and absence of a menace response. Cats with fore-
spp., FeLV, or astroviruses.14-17 Disease generally occurs after an brain disease may show abnormal behavior, such as aggression
incubation period of 2 to 10 days. The peracute form of disease or decreased mentation. Examination of the ocular fundus may
involves death without apparent premonitory signs. Infection of reveal folding of the retina, evidence of retinal degeneration
kittens or adult cats results in clinical signs of fever, lethargy, with discrete gray spots, and optic nerve hypoplasia. Retinal
vocalization, weakness, and inappetence, which may progress lesions may be an incidental finding in older, recovered cats,
to profound dehydration, vomiting, sometimes watery to hem- which includes surviving cats with cerebellar hypoplasia.
orrhagic diarrhea, and rapid loss of weight. Some cats develop
only anorexia and lethargy, in the absence of vomiting, diar- Diagnosis
rhea, or leukopenia.3 Secondary bacterial infections appear to be
essential for signs of disease to occur. Death ­usually results from Laboratory Abnormalities
complications relating to dehydration, electrolyte imbalances, Complete Blood Count
hypoglycemia, hemorrhage, or bacteremia and endotoxemia. The most common abnormality on the CBC in feline panleu-
In the developing fetus or neonate, FPV replicates in a vari- kopenia is leukopenia, which is due to a neutropenia and lym-
ety of tissues. Abortion, congenital abnormalities, or infertil- phopenia (Box 19-1).3 Total leukocyte counts may be as low
ity can result from infection early in pregnancy, although the as 50 cells/µL, and toxic band neutrophils may be present. In
queen is generally otherwise unaffected. Later in pregnancy or one study, only 65% of 187 cats with panleukopenia were leu-
in neonates up to approximately 1 week of age, viral destruc- kopenic, so an absence of leukopenia does not rule out FPV
tion of Purkinje cells and granule precursor cells located in the infection. Recovery may be associated with lymphocytosis and
cerebellar external granular layer leads to cerebellar hypoplasia
(Figure 19-1). The severity of infection can vary between kittens
in a litter. Sometimes a portion of the litter fails to survive and
the remainder develop neurologic signs.18 Signs of cerebellar
ataxia are nonprogressive and are most apparent when kittens
begin to walk at about 2 to 3 weeks of age, although menta-
tion and appetite are otherwise normal, and these kittens can
sometimes make acceptable pets. Other developmental central
nervous system (CNS) abnormalities have been reported less
commonly, and include hydrocephalus, porencephaly (cystic
lesions within the cerebral hemispheres), or hydranencephaly
(complete replacement of the cerebral hemispheres with cystic
lesions). These abnormalities may be accompanied by signs of
forebrain damage, such as seizures and behavioral changes.
Unlike canine parvoviruses, FPV appears to be able to infect
neurons other than the cerebellar Purkinje cells, which are ter-
minally differentiated cells.19 Ocular lesions can also develop
and include retinal folding, dysplasia and degeneration, and A
optic nerve hypoplasia.
The DNA of FPV has been detected in the myocardium of
cats with hypertrophic, dilated, and restrictive cardiomyopathy,
but was not detected in a subset of healthy control cats.20 Addi-
tional studies to assess the role of FPV in feline myocarditis and
cardiomyopathy are needed.
Fecal shedding of virus usually lasts for several days, and in
some cats, it may persist for up to 6 weeks. Kittens infected in
utero have been reported to develop immune tolerance to the
virus, with viral persistence in the kidneys and lungs for up to
1 year in the absence of shedding.

Physical Examination Findings

The most common physical examination findings are weakness,
lethargy, and dehydration. Fever (103°F to 107°F or 39.5°C to
42.5°C) may be present early in the course of illness. Pain may
be noted on palpation of the abdomen, or a hunched posture
may be present. The perianal region may be contaminated with
feces. Oral ulceration and mucosal pallor may be present in
severely affected cats, and rarely, bacteremia may be accompa-
nied by icterus. Terminally, affected cats may be hypothermic,
bradycardic, and comatose.
B
FIGURE 19-1  A, Severe cerebellar hypoplasia in a 1-year-old male neutered domes-
tic shorthair that was euthanized for cerebellar signs and seizures. The neurologic signs
had been present since the cat was found at approximately 4 months of age. There is a
paucity of neurons and glial cells in all layers. B, Normal feline cerebellum. Note the dra-
matically increased width of the molecular layer when compared to (A).
 CHAPTER 19  Feline Panleukopenia Virus Infection and Other Viral Enteritides 189

leukocytosis. Thrombocytopenia and mild anemia are also com-
mon. Thrombocytopenia may result from damage to the mar-
row or, possibly, disseminated intravascular coagulation (DIC).
BOX 19-1
Prevalence of Laboratory Abnormalities in Cats with
Panleukopenia
Serum Biochemical Tests
Serum biochemistry analysis may show hypoalbuminemia,
hypoglobulinemia, and/or hypocholesterolemia; electrolyte
abnormalities such as hyponatremia or hypernatremia, hypo-
chloremia, hyperkalemia, or, less commonly hypokalemia; and
acid-base abnormalities (see Box 19-1). In severely affected cats,
azotemia, increased serum activities of AST or ALT, or hyper-
bilirubinemia may be present. Hyperglycemia or hypoglycemia
may also be identified.

Diagnostic Imaging
Leukopenia: 122/187 (65%) Plain Radiography
Thrombocytopenia: 83/153 (54%) As in dogs with parvoviral enteritis, abdominal radiography
Anemia: 91/187 (48%) in cats with panleukopenia may show evidence of poor serosal
Neutropenia: 64/137 (47%) detail and a fluid- and gas-filled gastrointestinal tract.
Lymphopenia: 53/137 (39%)
Hypoalbuminemia: 45/101 (45%) MRI Findings
Hypochloremia: 40/112 (36%) MRI of cats with neurologic signs due to FPV may reveal evi-
Hyponatremia: 41/127 (32%) dence of cerebellar agenesis or hypoplasia. Rarely, hydrocepha-
Hypoproteinemia: 46/153 (30%) lus, porencephaly, or hydranencephaly can be detected.18
Hyperglycemia: 48/168 (29%)
Increased AST activity: 26/98 (27%) Microbiologic Tests
Hyperkalemia: 30/132 (23%) Diagnostic assays available for feline panleukopenia are listed
Increased BUN: 36/168 (21%) in Table 19-1.
Hyperbilirubinemia: 19/134 (14%)
Increased ALT activity: 18/135 (13%) Serologic Diagnosis
Increased creatinine: 12/155 (8%) Use of serology for diagnosis of feline panleukopenia is com-
Hypokalemia: 9/132 (7%) plicated by widespread exposure or immunization, so serologic
Hypernatremia: 8/127 (6%) assays that detect antibody against FPV are generally used to
Hypoglycemia: 10/168 (6%) assess the need for vaccination rather than for diagnosis. They
can also be used in outbreak situations in order to determine
Modified from Kruse BD, Unterer S, Horlacher K, et al. Prognos- which cats are at risk for development of disease and virus shed-
tic factors in cats with feline panleukopenia. J Vet Intern Med ding, and which cats are protected and therefore at low risk.
2010;24:1271-1276. The gold standard method for FPV serology is hemagglutination

TABLE 19-1
Diagnostic Assays Available for Feline Panleukopenia
Assay Specimen Type Target Performance
Canine parvovirus Feces Parvoviral antigen Sensitivity varies with the assay used and the timing
fecal antigen of specimen collection. False negatives are common,
ELISA but a positive result generally indicates infection.
Histopathology Usually necropsy Crypt necrosis with intra- Can be used for necropsy diagnosis.
specimens, especially nuclear inclusions; FPV
gastrointestinal tissues antigen with IHC or IFA
Polymerase chain Feces, tissue samples FPV DNA Sensitivity and specificity varies depending on assay
reaction (PCR) design. The extent to which attenuated live vaccine
virus can be detected after vaccination is not well
understood. Because of the high sensitivity of some
assays, the significance of a positive result may be dif-
ficult to interpret. False-negative results may occur as
a result of inhibition of PCR by components of feces.
Fecal electron Feces Virus particles Not widely available, turnaround time can be slow,
microscopy and may be expensive. Requires the presence of
large amounts of virus.
Virus isolation Feces, tissues FPV Difficult, not widely available. Used primarily as a
research tool.

FPV, feline panleukopenia virus; IFA, immunofluorescent antibody; IHC, immunohistochemistry.

190 SECTION 1  Viral Diseases
inhibition, which measures the ability of serum to prevent agglu-
tination of erythrocytes by the virus (see Chapter 2). Serum neu-
tralization assays may also be used. Point-of-care assays designed
to detect antibody titers to CPV have a low sensitivity (28%)
for detection of antibodies to FPV in cats.21 A point-of-care
assay designed to detect feline antibodies (ImmunoComb Feline
VacciCheck Test Kit, Biogal, Galed Labs, Israel) also had a low
sensitivity (49%), although specificity was high.21 The use of
this test for risk analysis in shelter situations may lead to inap-
propriate removal or isolation of cats with false-negative results
for protective antibody titers, which might waste time, space,
and financial resources.21 However, positive results should
­reliably indicate protection. The test can be performed quickly
(30 minutes) and requires as little as 5 µL of serum or plasma,
so provided it is understood that a negative result means either
a protected or susceptible status, and a positive result equates
to protection, the test still has the potential to provide useful
information.
Antigen Detection Enzyme Linked Immunosorbent Assay
FPV can be detected in feces or rectal swabs using antigen
assays designed to detect CPV.22,23 The sensitivity and speci-
ficity of these assays varies from one assay to another and with
the stage of infection, because virus shedding may be tran-
sient. In general, false-negative results are common with these
assays, but false positives are uncommon, so a positive test
result in a cat with consistent clinical signs suggests a diag-
nosis of feline panleukopenia. The specificity of one point-of-
care device (SNAP Parvo, IDEXX Laboratories, Westbrook,
ME) was high; 54 of 55 positive assay results were confirmed
with a PCR assay. Another study of 52 cats with diarrhea
and 148 healthy cats showed variability in the sensitivity
and specificity of five different test kits when compared with
fecal electron microscopy.23 Sensitivity ranged from 50% to
80%, and specificity ranged from 94% to 100%. This study
included only 10 cats with FPV as determined with electron
microscopy. Additional studies are warranted that evaluate
the sensitivity and specificity of these assays in larger numbers
of cats with FPV infection when both real-time PCR and elec-
tron microscopy are used as the gold standard. False-positive
fecal antigen assay results after vaccination with attenuated
live viral vaccines appear to be uncommon, but again vary
with the test used.24 Of the SNAP Parvo (IDEXX Laborato-
ries), AGEN CPV (AGEN Biomedical Ltd., Brisbane, Austra-
lia), and the Witness CPV (Synbiotics Corp, San Diego, CA),
the SNAP Parvo was least likely to yield positive results after
vaccination.
Fecal Electron Microscopy
Fecal electron microscopy is still offered by some institutions
for diagnosis of viral enteritis. It may also facilitate diagnosis
of other infections such as rotavirus, astrovirus, torovirus, and
coronavirus infections. Turnaround time may be slow. Gener-
ally speaking, large amounts of virus must be present for results
to be positive, and technical expertise is required to accurately
identify virus in the stool.
Virus Isolation
FPV can be isolated in feline cells, but as with CPV, isolation
can difficult, and the virus shows minimal cytopathic effects.
As a result, isolation of FPV is a specialized procedure that is
uncommonly used for diagnosis.
Molecular Diagnosis Using the Polymerase Chain Reaction
Specific real-time PCR assays have been developed for detection
of FPV and differentiation of FPV from CPV-2 variants and are
offered by veterinary diagnostic laboratories. Clinicians should
contact their laboratory to determine the specificity of the assay
offered.25 These assays can be used on whole blood or feces.
The extent to which these assays detect attenuated live vaccine
virus after vaccination requires further study. Assays have also
been developed that differentiate between field and vaccine
strains of FPV.26
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in feline panleukopenia include thymic
involution; thickening, distention, and discoloration of the intes-
tinal wall with serosal hemorrhage (Figure 19-2); and enlarged,
edematous mesenteric lymph nodes. The intestine may contain
bloody liquid contents, and mucosal hemorrhage may be identi-
fied. Hemorrhages may be visible on the surface of other organs
as well. In some cats, mild pleural or peritoneal effusion is pres-
ent. Cats infected prenatally may have cerebellar aplasia, or more
commonly a small cerebellum (often half to three-quarters normal
size).27 Rarely other developmental CNS abnormalities such as
hydrocephalus, hydranencephaly, or porencephaly are observed.18
Histopathologic Findings
Histopathologic findings in the intestinal tract are similar to those
described for CPV-2 infection, with crypt dilation and necrosis
of crypt epithelial cells, accumulation of cellular debris, neutro-
phil infiltration, loss of villi, and submucosal edema throughout
the small and large intestines; the jejunum and ileum are usually
most severely affected (Figure 19-3, A). Acutely affected cats
show widespread lymphoid depletion and there may be hyper-
plasia of mononuclear phagocytes. Intranuclear inclusions are
found in some cats (Figure 19-3, B). Examination of the bone
marrow may reveal bone marrow hypoplasia. Examination of
the cerebellum shows cellular depletion; reactive astrocytosis
may be present. Immunohistochemistry or immunofluorescent
FIGURE 19-2  Intestinal tract of a 2-year-old intact female domestic longhair cat
with severe feline panleukopenia. The intestinal loops are dilated and flaccid and discol-
ored red to purple. Ruler = 1 cm.
 CHAPTER 19  Feline Panleukopenia Virus Infection and Other Viral Enteritides 191

antibody may be used to document the presence of the virus
within tissues (see Figure 19-3, C).
Treatment and Prognosis
Antimicrobial Treatment and Supportive Care
Treatment of cats with feline panleukopenia is with supportive
care, especially intravenous crystalloids and parenteral antimi-
crobial drug treatment as for CPV-2 infections (see Chapter 14).
Dextrose supplementation of the fluids may be required, and
blood glucose concentration should be monitored. Oral intake
of food and water should be withheld until vomiting has ceased.
Experience with early enteral nutrition has not been reported
in cats, and extreme care is warranted to prevent aspiration
pneumonia. Antiemetics such as metoclopramide or ondanse-
tron may be effective. In contrast to canine parvoviral enteritis,
treatment with rfIFN-ω has not been beneficial for treatment of
feline panleukopenia, although increased antibody production
and a reduced acute inflammatory response was observed.28
Prognosis
Cats with panleukopenia that survive the first 5 days of treat-
ment usually recover, although recovery is often more pro-
longed than it is for dogs with parvoviral enteritis. In 244 cats
with feline panleukopenia from Europe, the survival rate was
51.1%.3 Nonsurvivors had lower leukocyte and platelet counts
than survivors, and cats with white cell counts below 1000/µL
were almost twice as likely to die than those with white cell
counts above 2500/µL. Only total leukopenia, and not lym-
phopenia, was correlated with mortality. Hypoalbuminemia
and hypokalemia were also associated with an increased risk of
mortality. In contrast to dogs with parvoviral enteritis, mortal-
ity in cats does not appear to be correlated with age.
Cerebellar signs in kittens with cerebellar hypoplasia typi-
cally do not progress and may improve slightly as a result of
compensatory responses from other senses such as vision.29
Immunity and Vaccination
Recovery from feline panleukopenia is thought to confer life-
long immunity. Effective vaccines are widely available, and
include both parenteral inactivated and attenuated live viral
vaccines. An intranasal FPV, FHV-1 and feline calicivirus vac-
cine is available; its use has been controversial, because panleu-
kopenia is a systemic disease. An outbreak of salmonellosis and
panleukopenia occurred in one cattery that used an intranasal
FPV vaccine.4 No difference was noted in seroconversion rates
between five cats vaccinated with the intranasal vaccine and five
cats vaccinated with a parenteral attenuated live vaccine, but
the number of cats in this study was small, and a larger number
of cats that were vaccinated with the parenteral vaccine had
protective antibody titers on day 7.30
Both inactivated and attenuated live vaccine types induce
protective antibody titers after vaccination in a high propor-
tion of cats,5 although the attenuated live vaccine may be
more likely to induce protective titers than the inactivated vac-
cine.24 Provided maternally-derived antibody (MDA) is absent,
immunity occurs within 1 week after a single vaccination with
an attenuated live viral vaccine31 and lasts for at least 3 years,
and possibly for life.32 Nevertheless, two doses, 3 to 4 weeks
apart, have been recommended for initial vaccination with
attenuated live vaccines in the absence of MDA. Two injec-
tions are always required for inactivated vaccines, and maxi-
mal immunity does not occur until 1 week after the second
dose. However, even with an inactivated vaccine, challenge
7.5 years after vaccination was associated with protection.33 The
use of inactivated vaccines should be reserved for immunosup-
pressed cats or colostrum-deprived neonates that are less than
4 weeks of age, or when there is a need to vaccinate pregnant cats.
In shelter situations, the use of attenuated live vaccines is always
recommended because of the slow onset of immunity with inac-
tivated vaccines.34 FPV vaccines protect cats against challenge
with CPV-2b strains.35,36 However, cross-reactivity of antibod-
ies induced by FPV vaccination to CPV-2 strains was lower than
that to FPV as determined using hemagglutination inhibition.37
The extent to which FPV vaccines protect against infection with
other CPV-2 variants requires further investigation.38
The most common reason for vaccine failure is interference
by MDA. Maternal antibody persists until at least 12 weeks,
and possibly longer in some cats. Virus neutralization titers
above 1:10 are likely to interfere with vaccination, and kittens
with titers below 1:40 are generally considered to be suscep-
tible to infection by FPV. Kittens should be vaccinated every
3 to 4 weeks from 6 to 8 weeks of age, and it is recommended

A B C
FIGURE 19-3  A, Histopathology of the jejunum from a kitten with panleukopenia. Villi are rounded and blunted with nearly complete epithelial loss and crypt dilation. H&E stain.
B, Histopathology of the jejunum from a young barn cat that died after several days of profound weakness and neurologic signs. Six other cats in the barn died with the same signs. Mucosal
crypts are dilated and contain debris, and intranuclear inclusions are present (small arrows). Another cell (large arrow) is foamy and degenerate and has a hyperchromatic nucleus. H&E
stain. C, Same cat as in B. The presence of FPV in the intestinal tract is confirmed with immunohistochemistry (brown stain). (Courtesy Dr. Patricia Pesavento, University of California, Davis
Veterinary Anatomic Pathology Service.)
192 SECTION 1  Viral Diseases
that the last vaccine in the kitten series be given no earlier
than 14 to 16 weeks of age. When there is a history of an out-
break situation, the final booster could be given no earlier than
18 to 20 weeks of age.39 In all situations, a booster should be
administered at 1 year, and every 3 years thereafter.
Vaccination of pregnant queens with attenuated live viral
vaccines can cause cerebellar hypoplasia or fetal losses. The fre-
quency with which this occurs is unknown. As a result, it has
been suggested that pregnant queens only be vaccinated with
attenuated live FPV vaccines if they are being introduced into
a shelter and quarantine while immunization with inactivated
vaccines is performed is not possible. Alternatively, assessment
for protective antibody titers with an in-house test kit (where
available) could be performed.
Prevention
New kittens should not be introduced into households that
previously contained cats infected with FPV unless they are
fully vaccinated. In the face of an outbreak, exposed and sus-
ceptible kittens may be effectively protected for 2 to 4 weeks
through subcutaneous or intraperitoneal administration of
CASE EXAMPLE
Signalment: “Callie”, a 2 year-old, female intact domestic
longhair from Woodland, CA
History: Callie was brought to an emergency clinic for acute
onset of collapse and severe illness. The current owner had
fostered the cat for 1 week after she was found as a stray. The
cat had been nursing a litter of kittens. The kittens were 4
weeks old, being weaned and apparently healthy. Since be-
ing fostered, the cat had exhibited a progressive decrease
in appetite and thirst, and her feces had become soft and
pasty. The night before she was brought to the emergency
clinic, she had been bathed, and afterwards she vomited
bile-stained fluid twice and was placed on a heating pad.
The following morning she was found laterally recumbent
and minimally responsive.
Physical Examination:
Body Weight: 2.3 kg
General: Stuporous mentation, estimated to be 8% to 10%
dehydrated, T < 92°F (<33°C), HR = 132 beats/min, RR = 32
breaths/min, mucous membranes pale and tacky, unable
to assess CRT. Fecal and urinary stains were present
around the perineum and on the caudal aspect of the
pelvic limbs.
Musculoskeletal: Body condition score 2/9. Diffuse muscle
wasting was present. The cat was laterally recumbent and
nonambulatory.
Cardiovascular: Weak femoral pulses. No murmurs or
arrhythmias were detected.
Gastrointestinal and Urogenital: The abdomen was soft
and nonpainful on palpation. Fluid-filled intestinal loops
were palpated, and the urinary bladder was small (<5 cm) and
soft.
All Other Systems: No abnormalities were detected.
2 mL of type-matched serum from cats with a high antibody
titer.39,40 However, this is only effective when administered
before the onset of clinical signs, and it can interfere with subse-
quent vaccination. For these kittens, it has been recommended
that vaccination be withheld for 3 weeks after the serum has
been administered.39 Passive immunization may be useful when
cats are introduced into a shelter situation where a known prob-
lem exists. Repeated treatment with serum should be avoided
because hypersensitivity reactions may occur. Prevention of
feline panleukopenia should also include proper disinfection
with disinfectants that are effective against parvoviruses, such
as bleach, accelerated hydrogen peroxide, or potassium peroxy-
monosulfate (see Chapter 11) and, in shelter situations, isola-
tion or removal of cats that develop gastrointestinal illness, and
separate housing for healthy kittens.
Public Health Aspects
Although FPV is not known to infect humans, a unique strain of
FPV was recently isolated from a diarrheic monkey in China.41
This strain was shown to cause panleukopenia in inoculated
cats.
Laboratory Findings:
CBC:
HCT 46% (30-50%)
MCV 49.8 fL (42-53 fL)
MCHC 30.4 g/dL (30-33.5 g/dL)
WBC 150 cells/µL (4500-14,000 cells/µL)
Neutrophils 0 cells/µL (2000-9000 cells/µL)
Lymphocytes 141 cells/µL (1000-7000 cells/µL)
Highly reactive lymphocytes 6 cells/µL
Monocytes 3 cells/µL (50-600 cells/µL)
Platelets 32,000 platelets/µL (180,000-500,000 platelets/µL).
Serum Chemistry Profile:
Sodium 141 mmol/L (151-158 mmol/L)
Potassium 4.0 mmol/L (3.6-4.9 mmol/L)
Chloride 111 mmol/L (117-126 mmol/L)
Bicarbonate 23 mmol/L (15-21 mmol/L)
Phosphorus 6.0 mg/dL (3.2-6.3 mg/dL)
Calcium 7.4 mg/dL (9.0-10.9 mg/dl)
BUN 24 mg/dL (18-33 mg/dL)
Creatinine 0.5 mg/dL (1.1-2.2 mg/dL)
Glucose 68 mg/dL (63-118 mg/dL)
Total protein 3.2 g/dL (6.6-8.4 g/dL)
Albumin 1.6 g/dL (2.2-4.6 g/dL)
Globulin 1.6 g/dL (2.8-5.4 g/dL)
ALT 125 U/L (27-101 U/L)
AST 143 U/L (17-58 U/L)
ALP 4 U/L (14-71 U/L)
Creatine kinase 2409 U/L (73-260 U/L)
Gamma GT <3 U/L (0-4 U/L)
Cholesterol 68 mg/dL (89-258 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL)
Magnesium 2.5 mg/dL (1.5-2.5 mg/dL).
Imaging: An abdominal ultrasound showed marked fluid dis-
tention of the small intestines.
Microbiologic Testing: In-clinic ELISA serology for FeLV anti-
gen and FIV antibody: negative.
 CHAPTER 19  Feline Panleukopenia Virus Infection and Other Viral Enteritides
Treatment and Outcome: Callie was treated with active
warming, and a central venous access line was placed. Intra-
venous crystalloids (four warmed 60-mL boluses of lactated
Ringer’s solution [LRS], each given over 20 minutes) were ad-
ministered, after which an venous acid-base panel showed
a pH of 7.267 (7.31-7.46), bicarbonate of 17.5 mmol/L (14-
22 mmol/L), base excess of −8.1 mmol/L (−4 to +2 mmol/L),
pCO2 of 39.7 mm Hg (25-37 mmHg), lactate of 4.5 mEq/L
(<2 mEq/L), glucose of 52 mg/dL, potassium of 3.1 mEq/L,
sodium of 141 mEq/L, and ionized calcium of 1.17 (1.1-1.4
mmol/L). A dextrose bolus and ticarcillin-clavulanic acid
(22 mg/kg, q6h, IV) were administered. The cat repeatedly
vomited blood-tinged fluid, and so treatment with meto-
clopramide (0.02 mg/kg/hr) and famotidine (0.5 mg/kg IV
q12h) was initiated. Aggressive crystalloid fluid therapy was
continued (LRS supplemented with 30 mEq/L KCl and 2.5%
dextrose), and treatment with hetastarch was also initiated.
Systolic blood pressure (Doppler) was 60 to 90 mm Hg, heart
rate increased to 170 beats/min, and rectal temperature in-
creased to 100°F. When the CBC results were available, the
cat was placed in the isolation ward.
Pasty diarrhea that contained sloughed mucosa occurred
every 1 to 2 hours, which transitioned to liquid red feces
over 24 hours. A CBC again showed absolute neutropenia
and 15,000 platelets/µL. After another 24 hours, the cat’s
condition deteriorated despite aggressive treatment and
monitoring. Partial parenteral nutrition was initiated. That
evening, pyrexia developed (104.6°F) as well as tachypnea
and increased respiratory effort. The owner elected eutha-
nasia. The cat regurgitated approximately 200 mL of dark
brown fluid at the time of euthanasia.
Gross Necropsy Findings: Ten mL of semiopaque red fluid
was present in the peritoneal cavity. Streaks of hemorrhage
were noted throughout the skeletal muscle. Between the
pylorus and the ileocecocolic junction, the serosa of the
small intestinal tract was dark red to purple to black (see
Figure 19-2). The entire small and large intestinal tract was
dilated and flaccid. The small intestinal mucosa was dark red
SUGGESTED READINGS
Kruse BD, Unterer S, Horlacher K, et al. Prognostic factors in cats with
feline panleukopenia. J Vet Intern Med. 2010;24:1271-1276.
Neuerer FF, Horlacher K, Truyen U, et al. Comparison of different in-
house test systems to detect parvovirus in faeces of cats. J Feline Med
Surg. 2008;10:247-251.
Truyen U, Addie D, Belak S, et al. Feline panleukopenia. ABCD guidelines
on prevention and management. J Feline Med Surg. 2009;11:538-546.
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193
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material. The mesenteric lymph nodes were prominent, and
their cut surface was discolored red to dark pink. The liver
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surface of the urinary bladder.
Histopathologic Findings: Within the duodenum, jejunum,
and ileum there was severe, subacute, diffuse necrotizing
and fibrinohemorrhagic enteritis. The villi were necrotic,
fused, and markedly blunted with a minimal inflammatory
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Diagnosis: Feline panleukopenia
Comments: The cat in this report was a stray with an un-
known vaccination history, which demonstrates that severe
disease can occur even in adult cats. Antemortem diagnos-
tic testing for FPV with a fecal antigen ELISA assay or PCR
assay was discussed with the owner, but was declined be-
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the lack of impact that a positive or negative result would
have had on the treatment plan. Secondary bacterial sepsis
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such as feline coronavirus, or enteropathogenic bacteria
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canine parvovirus types 2a and 2c in domestic cats. Clin Diagn Lab
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9. Ohshima T, Mochizuki M. Evidence for recombination between
feline panleukopenia virus and canine parvovirus type 2. J Vet Med
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10. Ikeda Y, Mochizuki M, Naito R, et  al. Predominance of canine
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11. Muir P, Harbour DA, Gruffydd-Jones TJ, et al. A clinical and micro-
biological study of cats with protruding nictitating membranes and
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13. Goodman LB, Lyi SM, Johnson NC, et al. Binding site on the trans-
ferrin receptor for the parvovirus capsid and effects of altered affinity
on cell uptake and infection. J Virol. 2010;84:4969-4978.
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15. Lutz H, Castelli I, Ehrensperger F, et al. Panleukopenia-like syndrome
of FeLV caused by co-infection with FeLV and feline panleukopenia
virus. Vet Immunol Immunopathol. 1995;46:21-33.
16. Ikegami T, Shirota K, Goto K, et al. Enterocolitis associated with
dual infection by Clostridium piliforme and feline panleukopenia
virus in three kittens. Vet Pathol. 1999;36:613-615.
17. Mochizuki M, Osawa N, Ishida T. Feline coronavirus participation
in diarrhea of cats. J Vet Med Sci. 1999;61:1071-1073.
18. Sharp NJ, Davis BJ, Guy JS, et al. Hydranencephaly and cerebel-
lar hypoplasia in two kittens attributed to intrauterine parvovirus
infection. J Comp Pathol. 1999;121:39-53.
19. Url A, Truyen U, Rebel-Bauder B, et al. Evidence of parvovirus replica-
tion in cerebral neurons of cats. J Clin Microbiol. 2003;41:3801-3805.
20. Meurs KM, Fox PR, Magnon AL, et  al. Molecular screening by
polymerase chain reaction detects panleukopenia virus DNA in
formalin-fixed hearts from cats with idiopathic cardiomyopathy
and myocarditis. Cardiovasc Pathol. 2000;9:119-126.
21. Digangi BA, Gray LK, Levy JK, et al. Detection of protective antibody
titers against feline panleukopenia virus, feline herpesvirus-1, and
feline calicivirus in shelter cats using a point-of-care ELISA. J Feline
Med Surg. 2011;13:912-918.
22. Abd-Eldaim M, Beall M, Kennedy M. Detection of feline panleu-
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23. Neuerer FF, Horlacher K, Truyen U, et al. Comparison of different
in-house test systems to detect parvovirus in faeces of cats. J Feline
Med Surg. 2008;10:247-251.
24. Patterson EV, Reese MJ, Tucker SJ, et  al. Effect of vaccination
on parvovirus antigen testing in kittens. J Am Vet Med Assoc.
2007;230:359-363.
25. Decaro N, Desario C, Lucente MS, et al. Specific identification of
feline panleukopenia virus and its rapid differentiation from canine
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2008;147:67-71.
26. Horiuchi M, Yuri K, Soma T, et al. Differentiation of vaccine virus
from field isolates of feline panleukopenia virus by polymerase
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27. De Lahunta A. Comments on cerebellar ataxia and its congenital
transmission in cats by feline panleukopenia virus. J Am Vet Med
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28. Paltrinieri S, Crippa A, Comerio T, et al. Evaluation of inflammation
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29. Penderis J. The wobbly cat. Diagnostic and therapeutic approach to
generalised ataxia. J Feline Med Surg. 2009;11:349-359.
30. Lappin MR, Veir J, Hawley J. Feline panleukopenia virus, feline
herpesvirus-1, and feline calicivirus antibody responses in serone-
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2009;11:159-162.
31. Jas D, Aeberle C, Lacombe V, et al. Onset of immunity in kittens
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2009;182:86-93.
32. Gore TC, Lakshmanan N, Williams JR, et al. Three-year duration
of immunity in cats following vaccination against feline rhinotra-
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33. Scott FW, Geissinger CM. Long-term immunity in cats vac-
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1999;60:652-658.
34. Day MJ, Horzinek MC, Schultz RD. WSAVA guidelines for the
vaccination of dogs and cats. J Small Anim Pract. 2010;51:1-32.
35. Gamoh K, Senda M, Inoue Y, et al. Efficacy of an inactivated feline
panleucopenia virus vaccine against a canine parvovirus isolated
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36. Chalmers WS, Truyen U, Greenwood NM, et al. Efficacy of feline
panleucopenia vaccine to prevent infection with an isolate of
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of cross-reactivity of virus neutralising antibodies induced by
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guidelines on prevention and management. J Feline Med Surg.
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to correct failure of passive transfer in kittens. J Am Vet Med Assoc.
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41. Yang S, Wang S, Feng H, et  al. Isolation and characterization of
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2010;143:155-159.
CHAPTER 20
Feline Coronavirus Infection
Jane E. Sykes
Overview of Feline Coronavirus Infections
First Described: 19631; a viral etiology was not identified until
the 1970s.
Cause: Feline coronavirus (Family Coronaviridae, genus
Coronavirus)
Affected Hosts: Cats and wild felids, especially cheetahs
Mode of Transmission: Fecal-oral
Geographic Distribution: Worldwide
Major Clinical Signs: Fever, lethargy, inappetence, vomiting,
diarrhea, dehydration, icterus, tachypnea, uveitis, neuro-
logic signs, abdominal distention due to ascites.
Differential Diagnoses: Toxoplasmosis, congestive heart
failure, carcinomatosis, lymphoma, pancreatitis, rabies,
cryptococcosis, bacterial peritonitis, pyothorax, bacterial
meningitis, chronic stomatitis, multiple myeloma, infec-
tion with FeLV or FIV.
Human Health Significance: Feline coronaviruses do not
infect humans.
Etiology and Epidemiology
Coronaviruses are large, enveloped, single-stranded RNA
viruses with club-shaped spikes on their outer surface (see
­Figure 14-1, B). They have the largest RNA genomes of all
known viruses. Feline coronaviruses (FCoV), like canine enteric
coronavirus, belong to the Group 1a coronaviruses (see Box
17-1). In fact, even canine enteric coronavirus has the poten-
tial to infect cats and cause diseases similar to those caused by
FCoV.2 Among FCoVs, there are two different serotypes, type
I and type II, which use different receptors for cellular entry
in  vitro3,4 but cause the same clinical manifestations. Type I
strains predominate worldwide.5-7 Type II strains, which are
thought to have evolved from genetic recombination between
canine enteric coronavirus and FCoV, are more readily grown
in culture and so have been more extensively studied; they
possess a spike protein that resembles that of canine enteric
coronavirus.
FCoVs cause enteric disease in cats as well as feline infec-
tious peritonitis (FIP), a serious systemic pyogranulomatous to
granulomatous disease that progresses over a period of weeks to
months and, once it occurs, is ultimately always fatal. FIP is a
major cause of death in young and young adult cats, especially
cats from multicat environments such as purebred catteries
and shelters. Wild cats, especially cheetahs, are also suscepti-
ble.8 The vast majority of domestic cats that develop FIP are
3 months to 3 years of age, with at least 50% of affected cats
aged 12 months or younger (Figure 20-1). However, FIP can
occur at any age, and there is a secondary peak of incidence
in geriatric cats (>10 years of age), possibly as a result of sub-
optimal immune function. Males and sexually intact cats have
been predisposed in some studies,9-11 and a disease peak may
exist in the fall and winter.12 Although the disease occurs in
all breeds, purebred cats are more susceptible; Abyssinians,
Australian mist, Bengals, birmans, Burmese, British shorthairs,
Himalayans, ragdolls, rexes, and possibly Scottish folds may
be predisposed.9-14 Breed predispositions may vary geographi-
cally and temporally depending on the preferences of breeders
in a region, and specific lines may be more predisposed than the
breeds themselves.6 The molecular basis of genetic susceptibility
to FIP is currently unclear. Siblings of cats that die of FIP may
be at increased risk for FIP.15
In multiple-cat household situations, cats are repeatedly
infected, shed virus, and recover, but some cats remain persis-
tently infected and chronically shed FCoV in the absence of clini-
cal signs (Figure 20-2). More than half, and as many as 100% of
cats in environments with more than six cats, become infected
with FCoVs.6 The seroprevalence is lower in cats from single-cat
households and among feral cats.16 However, even though the
prevalence of infection in multicat households is high, fewer than
10% of cats from large, multicat households ultimately develop
FIP. Thus, although the incidence of infection is high, the inci-
dence of disease in single- or two-cat households is only around
1 in 5000; in catteries it is around 5% to 10%.17,18 Provided
they are unrelated by birth, cats in households with a history of
FIP are not more likely to develop FIP than cats in households
without FIP.19 Thus, FIP is usually a sporadic disease that does
not spread from one cat to another. However, every few years,
epidemics of disease can occur in catteries or shelters, with mor-
tality rates that exceed 10%.12 Because it is an enveloped virus,
FCoV is readily inactivated by disinfectants and generally sur-
vives less than a day or two at room temperature. However, the
possibility of prolonged survival (up to 7 weeks) in the environ-
ment under certain conditions has been suggested.15,20 In this
situation, fomites might play an important role in transmission.
The epidemiology and pathogenesis of FIP has both fascinated
and confused veterinary virologists worldwide for decades. The
most widely accepted theory (the “internal mutation hypothe-
sis”) is that cats are initially infected with a low-pathogenicity
coronavirus after oronasal exposure, which results either in no
signs, or mild enteric disease. This low-pathogenicity virus has
been referred to as feline enteric coronavirus in some publica-
tions in order to distinguish it from virulent FIP virus. The use
of this name has been controversial, because although the virus
is primarily confined to the gastrointestinal tract (and especially
colonic epithelial cells), FCoV RNA can also be found in blood
195
196 SECTION 1  Viral Diseases
30
Number of cats
20
10
0
3-6 7-12 13-24 25-36 37-60 61-120 120-180 >180
Age (months)
FIGURE 20-1  Age distribution of 99 cats with necropsy-confirmed FIP at the UC
Davis VMTH. An additional six cats were reported to be “juvenile” or kittens. There were 38
females (17 intact) and 66 males (22 intact).
Overcrowded environment
Stress, fecal contamination
Genetically-predisposed Chronic coronavirus
cats shedders
FIGURE 20-2  Interplay between genetics, virus shedding, and environment in feline
coronavirus infections and FIP.
and tissue macrophages of cats that do not have FIP.21,22 In some
infected cats, the low-pathogenicity virus is believed to mutate to
a virulent strain that can multiply within macrophages without
hindrance by the immune system and incite a systemic pyogranu-
lomatous vasculitis. The mutation may occur shortly after initial
infection, or years later, which may explain why some indoor cats
from single-cat households develop FIP several years after they
are acquired. Virulent strains may not be able to replicate effec-
tively within the gut,23 which may be the reason why cat-to-cat
transmission of FIP does not occur, yet the disease can be trans-
mitted effectively by inoculating naïve cats with effusion from
a cat with FIP. Factors that contribute to immunosuppression,
such as concurrent viral infection, stress due to overcrowding,
surgery, or transport, and especially genetic factors may allow
viral replication and mutation to proceed unchecked. Simulta-
neous immune compromise of a large number of cats, such as
in a shelter situation, may explain epidemics of FIP. Other risk
factors for FIP include regular introduction of new cats to a cat-
tery and the proportion of cats in a cattery that shed coronavirus
chronically.12 There is no distinct mutation that allows avirulent
FCoV strains to be differentiated from virulent strains, and there-
fore no diagnostic test exists that distinguishes FIP from benign
FCoV strains. However, mutations in the spike protein gene,24,25
membrane protein gene,26 and the nonstructural 3c and 7b
genes23,27-29 may play a role. In particular, the 3c gene appears to
be disrupted in many (but not all) virulent FCoV strains.
The other hypothesis proposed to explain the pathogenesis
of FIP is that distinct circulating virulent and avirulent FCoV
strains exist, and the combination of infection with a virulent
FCoV and an individual cat’s genetic and environmental pre-
dispositions leads to FIP.25 It has also been suggested that both
hypotheses may play a role.30
Clinical Features
Signs and Their Pathogenesis
Cats are usually infected with FCoV by oronasal exposure
to virus in feces or fomites contaminated with fecal material.
Shared litter boxes are thought to play a major role in transmis-
sion.15 Replication of low-pathogenicity strains of FCoV in epi-
thelial cells at the tips of intestinal villi may be associated with
no signs, or acute or chronic, persistent or intermittent small-
bowel diarrhea, and less commonly, vomiting and/or inappe-
tence. Transient upper respiratory signs have been reported in
some cats on initial infection with FCoV.15 Virus is shed in the
feces from 1 week after infection. Some cats then shed large
quantities of virus continuously for life.12,22,31-33
Both serotype I and serotype II strains appear to enter macro-
phages via a lectin receptor known as fDC-SIGN (feline dendritic
cell-specific intercellular adhesion molecule grabbing non-inte-
grin receptor).3,34 Replication of virulent FCoV strains within
macrophages results in two forms of disease, which reflect the
immune response mounted by the host. FIP is an immune com-
plex disease. Noneffusive (“dry”) FIP occurs in cats that mount
a partial CMI response and is characterized by pyogranuloma-
tous to granulomatous inflammation within a variety of organs,
but especially the mesenteric lymph nodes, kidneys, liver, lungs,
brain, and eye. Solitary or multifocal granulomas of the intesti-
nal wall also occasionally develop, especially in the region of the
ileocecal junction (Figure 20-3).15 Effusive (“wet”) FIP occurs in
cats that are unable to mount an immune response and is char-
acterized by accumulation of high protein exudates in the thorax
and/or abdomen, which typically contain low numbers of cells.
Production of vascular endothelial growth factor by infected
monocytes may be lead to increased vascular permeability and
contribute to cavitary effusion.35 Many cats have a mixture of
both forms of the disease, and noneffusive disease may prog-
ress to effusive disease. Infection itself results in immune dys-
regulation, with a profound, virus-induced depletion of CD4+
and CD8+ cells; production of TNF-α, granulocyte-macrophage
colony stimulating factor (GM-CSF), and granulocyte colony-
stimulating factor (G-CSF) by infected macrophages; impaired
IFN-γ production; and hypergammaglobulinemia.36-38 The
mechanism of T-cell depletion is not clear, as the virus does not
infect lymphocytes, only monocytes and macrophages. Infection
of antigen-presenting cells, specifically dendritic cells, has been
hypothesized to lead to T-cell apoptosis. Progressive immune
system failure may be associated with a conversion to predomi-
nantly effusive disease manifestations. Despite the profound
T-cell deficiency that accompanies FIP, opportunistic infections
are rarely reported. Nevertheless, concurrent infections with
retroviruses and Toxoplasma gondii and opportunistic bacterial
infections can occur;6 the author is aware of one cat that was
co-infected with Sporothrix schenckii.

FIGURE 20-3  Colonic mass removed at surgery from a 7-month-old female spayed
domestic shorthair with anorexia and hematochezia. Histopathology showed severe, mul-
tifocal coalescing pyogranulomatous colitis and lymphadenitis. (Courtesy of the University
of California, Davis Veterinary Anatomic Pathology service.)
FIGURE 20-4  Obstructive hydrocephalus in an 8-month-old male neutered exotic
shorthair cat that developed ataxia and head tremors. Hydrocephalus and secondary
cerebellar herniation were found at necropsy. Histopathology revealed severe, multifocal
pyogranulomatous meningoencephalitis, choroiditis, and ventriculitis, and pyogranulo-
matous inflammatory lesions were also found throughout the thoracic and abdominal
viscera. (Courtesy of the University of California, Davis Veterinary Anatomic Pathology
service.)
The incubation period for FIP is highly variable. Kittens usu-
ally become infected at 4 to 8 weeks of age, when maternal
antibody begins to wane, but infections have been reported in
kittens as young as 2 weeks of age.20 Disease may occur a few
weeks after infection or years later, but most often it occurs 6
to 18 months after initial infection.19 Even after the onset of
systemic pyogranulomatous inflammatory disease, clinical signs
may not be apparent for months. In support of this, lesions
consistent with FIP have been found incidentally in cats during
abdominal surgery such as ovariohysterectomy.20
The clinical signs of FIP often change over time and depend
on the organs affected and the relative predominance of inflam-
matory versus effusive disease manifestations. The most com-
mon signs are lethargy and inappetence, as well as a fluctuating
fever that does not respond to antibacterial drug treatment.
Nevertheless, many cats are bright, appetent, and in good body
condition early in the course of illness. Some cats have increased
thirst and urination, possibly secondary to pyrexia. Ultimately,
weight loss develops, but owners of cats that develop abdomi-
nal distention may mistake the distention for weight gain or
pregnancy. Stunted growth may occur in affected kittens. Pleu-
ral effusion may be associated with tachypnea and respiratory
distress. Testicular enlargement may occur in cats with serositis
that involves the tunica vaginalis. FIP is responsible for approxi-
mately 10% of pericardial effusions in cats, the third most com-
mon cause of pericardial effusion after cardiomyopathy and
CHAPTER 20  Feline Coronavirus Infection 197
FIGURE 20-5  Keratic precipitates in a 5-year-old intact male Burmese cat with FIP.
(Courtesy of the University of California, Davis Veterinary Ophthalmology service.)
neoplasia.39 Rarely, pericardial effusion results in cardiac tam-
ponade. Pyogranulomatous or granulomatous inflammation
may lead to mesenteric lymphadenomegaly, irregular renomeg-
aly, intestinal masses, hepatomegaly, icterus, pneumonia, uve-
itis, chorioretinitis, and, rarely, nodular skin lesions. Neurologic
signs, which can include focal or generalized seizures, occur in
at least 10% of cats with FIP and result primarily from menin-
goencephalitis, meningomyelitis, ependymitis, choroiditis, and
obstructive hydrocephalus. Obstructive hydrocephalus occurs
secondary to choroiditis and ependymitis (Figure 20-4). In one
study, FIP was responsible for almost half of all neurologic
disease in 97 cats due to infectious or inflammatory causes.40
Occasionally profound anemia occurs secondary to immune-
mediated hemolysis13,14 or possibly microangiopathic damage,
whereby erythrocytes are lysed as they travel through inflamed
blood vessels. Immune-mediated glomerulonephritis has also
been reported, and FIP should always be considered in cats with
protein-losing nephropathy, which is otherwise rare in cats.41
Uncommonly, lameness occurs as a result of synovitis.6
Physical Examination Findings
Physical examination findings in cats with FIP reflect the type
of disease present (effusive versus noneffusive) and the loca-
tion where lesions occur. Cats with respiratory tract involve-
ment may show tachypnea, and if there is pleural effusion, a
rapid, shallow breathing pattern and muffled heart and lung
sounds may be present. Other signs include pyrexia, dehydra-
tion, mucosal pallor or icterus, a thin body condition, and
evidence of ascites. Abdominal palpation may reveal hepato-
megaly, irregular renomegaly, and/or abdominal mass lesions
that result from mesenteric lymphadenomegaly or intestinal
pyogranulomas. Sometimes pain is appreciated on abdominal
palpation, which may reflect pancreatic involvement in some
cats. Testicular enlargement may be detected in intact male
cats. A wide range of neurologic signs may be present, such as
obtundation, twitching, tremors, behavioral changes, nystag-
mus, hyperesthesia, exaggerated segmental reflexes, ataxia, uri-
nary incontinence, or cranial nerve defects. Ocular signs include
conjunctivitis, mucopurulent ocular discharge, thickening and
hyperemia of the nictitans, uveitis with dyscoria or anisoco-
ria, aqueous flare, keratic precipitates, hypopyon, hyphema,
chorioretinitis, perivascular infiltrates, retinal detachment, or
blindness (Figure 20-5).
198 SECTION 1  Viral Diseases

TABLE 20-1
Complete Blood Count Findings at Admission in 38 Cats with Necropsy-Confirmed Feline Infectious Peritonitis
at the UC Davis VMTH
Percent below Percent within Percent above Range for
Reference the Reference the Reference the Reference Cats with Number
Test Range Range Range Range FIP Tested
Hematocrit (%) 30-50 68 32 0 17-53 38
MCV (fL) 65-75 18 82 0 36-52 38
MCHC (g/dL) 33-36 5 58 37 28-36 38
RDW (%) 14-18 0 30 70 14-33 27
Neutrophils* 2000-9000 3 26 71 416-49,313 38
(cells/µL)
Band neutrophils* 0-rare 0 50 50 0-3251 38
(cells/µL)
Metamyelocytes 0 0 95 5 0-276 38
(cells/µL)
Monocytes 50-600 5 71 24 0-820 38
(cells/µL)
Lymphocytes 1000-7000 58 42 0 89-6886 38
(cells/µL)
Eosinophils 150-1100 71 29 0 0-770 38
(cells/µL)
Platelets 180,000-500,000 37 44 19 30,000-874,000 27†
(cells/µL)

FIP, Feline infectious peritonitis; RDW, red cell distribution width.

*22 (58%) had evidence of toxic neutrophils.
†A smear was evaluated manually for 37 of the 38 cats. The presence of macroplatelets were reported for 18 (49%) of cats.

Diagnosis regenerative or nonregenerative (Table 20-1). Microcytosis may

Currently, definitive diagnosis of FIP is made only by immunohis-
tochemical staining for coronavirus antigen within lesions charac-
terized by pyogranulomatous or granulomatous vasculitis. Because
it can be difficult or impossible to safely obtain biopsy specimens
from cats with FIP, antemortem diagnosis is often only suspected
on the basis of history, signalment, and clinical and laboratory
findings, and by ruling out other causes of disease. Provided it is
correctly performed and interpreted, immunocytochemistry may
be helpful. Because the presence of the characteristic effusion is
most helpful for antemortem diagnosis, efforts should be always
made to identify and analyze any fluid that is present in body cavi-
ties. When owner funds are limited, laboratory analysis of effu-
sion, rather than blood, may be the most economic diagnostic
approach. Unfortunately, the lack of a definitive noninvasive diag-
nostic assay for FIP and the extremely poor prognosis sometimes
leads clinicians to perform large numbers of diagnostic tests in the
hope that an answer will appear. In other situations, the diagnosis
of FIP is made too hastily, and euthanasia is performed without
sufficient clinical and laboratory justification.
Laboratory Abnormalities
Complete Blood Count
A mild, nonregenerative anemia is often present in cats with FIP,
and sometimes severe anemia occurs, which is usually poorly
be present. Examination of erythrocyte morphology occasion-
ally reveals schistocytosis, mild normoblastosis, or agglutina-
tion. There may be a leukocytosis due to a neutrophilia and
monocytosis, or leukopenia. Lymphopenia occurs in more than
50% of affected cats, and eosinopenia is also common. In some
cats, a left shift and evidence of toxic neutrophils are seen. Mild
to moderate thrombocytopenia is common in cats with noneffu-
sive disease and may reflect the presence of disseminated intra-
vascular coagulation or immune-mediated platelet destruction.
However, thrombocytosis can also occur.
Serum Biochemical Tests
Many cats with FIP have hyperproteinemia due to hyper-
globulinemia, which results from a polyclonal gammopa-
thy (Figure 20-6). Rarely, a monoclonal gammopathy can
occur.42 Total protein concentrations may be as high as 12 g/
dL (Table 20-2).20 In one study, hyperglobulinemia was pres-
ent in 50% of cats with effusion and 70% of cats without
effusion.43 Globulin concentration may decrease terminally,
so cats with advanced disease may have protein concentra-
tions that are within the reference range.14 Hypoalbumin-
emia is often present because of liver involvement, leakage
from damaged vessels, urinary loss in cats with glomerulone-
phritis, or inflammation (albumin is a negative acute-phase
reactant protein). Thus, the serum albumin:globulin ratio
 CHAPTER 20  Feline Coronavirus Infection 199

A B
FIGURE 20-6  A, Densitometric scan of serum protein electrophoresis of normal feline serum. B, Scan from a 9-month-old male neutered domestic shorthair cat with FIP. There is a
polyclonal gammopathy, represented by a broad peak in the γ-globulin region, with a mild decrease in the albumin and mild increases in the α2 and β1 fractions. (A redrawn from Baker
RJ, Valli VE. Electrophoretic and immunoelectrophoretic analysis of feline serum proteins. Am J Vet Res 1988;52[3]:308-304.)

TABLE 20-2
Findings on Serum Biochemistry Analysis in 36 Cats with Necropsy-Confirmed Feline Infectious Peritonitis
at the UC Davis VMTH
Percent below Percent within Percent above
Reference the Reference the Reference the Reference Range for Number of
Test Range Range Range Range cats with FIP Cats Tested
Sodium (mmol/L) 151-158 94 6 0 129-152 35
Potassium (mmol/L) 3.6-4.9 26 74 0 2.2-5.4 35
Chloride (mmol/L) 117-126 91 9 0 94-121 35
Bicarbonate (mmol/L) 15-21 8 75 17 12-25 36
Calcium (mg/dL) 9.0-10.9 56 44 0 6.6-10.6 36
Phosphorus (mg/dL) 3.2-6.3 8 69 22 1.9-8.4 36
Creatinine (mg/dL) 1.1-2.2 69 28 3 0.4-2.8 36
BUN (mg/dL) 18-33 56 31 14 10-58 36
Glucose (mg/dL) 63-118 0 47 53 63-381 36
Total protein (g/dL) 6.6-8.4 31 28 42 4.1-11.9 36
Albumin (g/dL) 2.2-4.6 50 50 0 0.9-3.6 36
Globulin (g/dL) 2.8-5.4 3 42 56 2.5-9.4 36
Cholesterol (mg/dL) 89-258 17 83 0 56-247 36
Total bilirubin (mg/dL) 0-0.2 0 40 60 0-5.3 36
ALT (U/L) 27-101 50 25 25 18-648 36
AST (U/L) 17-58 6 43 51 0-1554 36
ALP (U/L) 14-71 28 61 11 0-161 36
GGT (U/L) 0-4 0 96 4 0-5 23

FIP, feline infectious peritonitis.

may be more useful than the globulin alone for diagnosis;
ratios less than 0.8 are uncommon (but not impossible) in
cats with FIP, so they help to rule out (but not to rule in) a
diagnosis of FIP.44,45 Other variable findings include hypona-
tremia, hypokalemia, hypochloremia, hyperglycemia, azote-
mia, increased liver enzyme activities, hypocholesterolemia,
and hyperbilirubinemia. The cause of hyperbilirubinemia is
not clear, but it may result from hemolysis, hepatic necrosis,
and/or cholestasis.
Measurement of α1-acid glycoprotein (an acute phase pro-
tein) has been suggested for diagnosis, because serum con-
centrations often exceed 1500 µg/mL in cats with FIP.20,46,47
However, α1-acid glycoprotein concentrations also increase
with other inflammatory diseases.20
200 SECTION 1  Viral Diseases

TABLE 20-3 TABLE 20-4

Composition of Body Cavity Effusions from 21 Cats with Composition of Cerebrospinal Fluid from 10 Cats with Necropsy-
Necropsy-Confirmed Feline Infectious Peritonitis at the Confirmed Feline Infectious Peritonitis at the UC Davis VMTH
UC Davis VMTH
Number
Number Reference of Cats
of Cats Test Range Median Range Tested
Test Range Mean ± SD Tested Total protein 44-4079 639 <25 4*
Total protein (g/dL) 2.9-8.1 5.1 ± 1.8 21 (mg/dL)
RBC (cells/µL) <100-38,600 ND 18 RBC (cells/µL) 3-850 340 0 9
TNC (cells/µL) 200-13,200 3683 ± 3474 19 TNC (cells/µL) 26-2637 303 0-2 10
Neutrophils (%) 3-97 61 ± 28 21 Neutrophils (%) 4-90 73 10
Lymphocytes (%) 0-22 6±6 21 Lymphocytes (%) 5-89 19 10
Monocytes (%) 1-96 33 ± 26 21 Monocytes (%) 0-20 7 10

Nineteen specimens were abdominal and two were pleural effusions. TNC, total neutrophil count.
ND, Not determined; SD, standard deviation; TNC, total neutrophil *Insufficient quantity available from some cats for determination of
count. protein concentration.

Urinalysis
The urinalysis in cats with FIP may be unremarkable or contain
protein due to glomerular or tubular damage. Hematuria and,
less commonly, pyuria and cylindruria may be present. Bilirubi-
nuria may be detected in cats with liver injury.
younger cats, the positive predictive value of the test is higher,
because diseases such as lymphoma and bacterial peritonitis are
less common. Positive test results indicate only the presence of
an exudate, so cytologic examination of the fluid must still be
performed.

Coagulation Profile Cerebrospinal Fluid Analysis

In addition to thrombocytopenia, abnormalities of coagula-
tion in cats with FIP include prolonged prothrombin time
and partial thromboplastin time as a result of severe liver
injury, and increased fibrin degradation product or D-dimer
concentrations.15
Analysis of Effusion Fluid
The “classic” FIP effusion fluid is a high-protein (greater than
3.5 g/dL) exudate that contains a low number of nucleated
cells (<5000 cells/µL), usually nondegenerate to mildly degen-
erate neutrophils and macrophages (Table 20-3). Erythropha-
gocytosis, leukophagia, and reactive mesothelial cells can be
observed in the fluid from some cats. Grossly, the fluid has a
yellow appearance and may contain fibrin clots. However, the
total protein content and cell counts of abdominal and pleural
effusions vary considerably, which complicates the diagnosis for
some cats with effusive disease. Very rarely, chylous effusions
occur.48 An effusion albumin/globulin ratio below 0.4 is sug-
gestive of FIP.49
The Rivalta test is a simple test that can differentiate between
transudates and exudates. In this test, a drop of 98% glacial
acetic acid is mixed with 7 to 8 mL of distilled water in a trans-
parent 10-mL tube. A drop of effusion is then added to the tube,
and if it dissipates in the solution, the test is negative. If it retains
its shape, stays attached to the surface, or moves slowly down
in the solution, then the test is positive.20 In a study of cats
with effusion, 35% of which had FIP and a conclusive Rivalta
test, the positive predictive value of this test for the diagnosis
of FIP was 58% (58% chance that a cat that tests positive truly
has FIP), and the negative predictive value was 93% (93%
chance that a cat that tests negative does not have FIP).50 In
The cerebrospinal fluid (CSF) of cats with neurologic FIP often
has increased protein content (30 to more than 1000 mg/dL, ref-
erence range less than 25 mg/dL) and increased total nucleated
cell count (20 to 10,000 cells/µL), usually consisting of a mixed
but predominantly neutrophilic cellular pleocytosis (Table
20-4). In some cats, protein content and leukocyte counts are
normal.51,52
Diagnostic Imaging
Plain Radiography
Plain thoracic radiography may reveal pleural effusion, enlarge-
ment of the cardiac silhouette in cats with pericardial effusion,
and pulmonary nodular or peribronchial infiltrates in cats with
pyogranulomatous pneumonia (Figure 20-7). Abdominal radio-
graphs may show loss of peritoneal or retroperitoneal detail due
to peritoneal effusion, hepatomegaly, splenomegaly, renomeg-
aly, or mass lesions associated with the gastrointestinal tract or
abdominal lymph nodes.
Sonographic Findings
Abdominal ultrasound findings in FIP include the presence of
anechoic or mildly echogenic peritoneal fluid; hyperechogenicity
and “clumping” of the mesentery; enlarged and hypoechoic
abdominal lymph nodes (Figure 20-8, A); enlargement and dif-
fuse or focal hypoechogenicity of the liver and spleen;53 renal
asymmetry with increased cortical echogenicity, hypoechoic
nodules, subcapsular fluid accumulation, or loss of corticome-
dullary distinction (see Figure 20-8, B); and/or thickening of all
intestinal wall layers or intestinal mass lesions. Pleural effusion
or comet-tail artifacts (due to pulmonary infiltrates) may be seen
through the diaphragm.

FIGURE 20-7  Lateral thoracic radiograph from a 9-month-old male neutered
domestic shorthair cat with FIP and pyogranulomatous pneumonia. There is a severe, dif-
fuse, patchy alveolar and nodular interstitial pattern with thickening of the bronchial walls
and mild pleural effusion.
Magnetic Resonance Imaging of the Central Nervous System
Findings on MRI that suggest FIP consist of ventricular dilata-
tion and variable contrast enhancement of the periventricular
regions, choroid, and meninges. In some cats, MRI findings are
unremarkable.
Microbiologic Tests
Serologic Diagnosis
Detection of antibodies to FCoV can be performed using immu-
nofluorescent antibody testing, ELISA, or virus neutralization.54
The methods used, as well as the titers themselves, vary con-
siderably between laboratories. For example, some laboratories
use related coronaviruses as a source of antigen for the test,
rather than FCoV.15 Use of a reliable laboratory that reports
quantitative titers (to the endpoint dilution, as well as down
to 1:100) is critical. Even when performed correctly, a posi-
tive FCoV antibody titer is not diagnostic for FIP, because cats
that have been exposed to avirulent FCoV strains or even other
related coronaviruses are also seropositive. Therefore, serology
is a “coronavirus antibody test” and not an “FIP test.” It has
been suggested that more cats have been killed as a result of
misinterpretation of FCoV antibody tests than by the disease
itself.20 Certainly a diagnosis of FIP should never be made based
on the presence of nonspecific clinical or laboratory abnor-
malities such as fever or leukocytosis and a positive coronavi-
rus antibody test. Occasionally (up to 10% of the time), cats
with advanced disease are seronegative, because of failure of
antibody production with severe immunosuppression, or the
complexing of antibody by the large quantities of virus pres-
ent. In one study, titers of 1:1600 or higher were highly sugges-
tive (94% chance) of FIP in the presence of compatible clinical
signs.44 In addition, strong positive titers (e.g., ≥ 1:6400) in cats
with consistent signs and laboratory abnormalities support a
diagnosis of FIP if a cat resides in a household that contains
only one or two cats, because cats often become seronegative
within a few months once they are removed from households
that contain large numbers of cats.
Other body fluids can also be analyzed for antibodies to
FCoV. In one study, positive antibody titers in effusion had
a positive predictive value of 90% and a negative predictive
value of 79%, but the magnitude of the titer did not corre-
late with the diagnosis of FIP.44 The presence of anti-FCoV
CHAPTER 20  Feline Coronavirus Infection 201
A
B
FIGURE 20-8  A, Abdominal ultrasound image from a 9-month-old male neutered
domestic shorthair cat with FIP and ileocecocolic lymphadenopathy. The lymph nodes are
enlarged and hypoechoic. B, Abdominal ultrasound image from a 1-year-old male intact
Scottish fold with FIP. There is renal irregularity and subcapsular fluid, as well as moderate
peritoneal effusion.
antibody in the CSF correlated well with a diagnosis of FIP in
one study,52 but not in another study.51 In addition, the pres-
ence of sufficient quantities of CSF for serology are frequently
not available.
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time reverse transcriptase–PCR (RT-PCR) assays have
been developed for detection of FCoV, but these do not differ-
entiate between virulent and avirulent strains. In addition, avir-
ulent strains can be found in the blood and tissues of cats that
do not have FIP,21,22 so the finding of virus in locations other
than the gastrointestinal tract is not helpful for diagnosis. False-
negative test results can occur when there are low quantities
of virus present or if degradation of RNA occurs during speci-
men transport. Some RT-PCR assays do not detect all strains of
FCoV. Positive RT-PCR results in blood or effusion fluid from
cats with other clinical abnormalities that suggest FIP do indi-
cate the presence of a coronavirus and, in that respect, may help
to support the diagnosis made, provided the limitations of the
test are recognized.
Immunostaining of FCoV Antigen
FCoV antigen can be detected in macrophages with immu-
nocytochemistry or immunohistochemistry (Figure 20-9).
Either fluorescent antibody or immunoperoxidase methods
202 SECTION 1  Viral Diseases

B
FIGURE 20-9  A, Cytospin preparation of a tracheobronchial lavage from the cat in Figure 20-7. The specimen was highly cellular (4400 cells/µL) and the cells consisted of 42% degen-
erate and nondegenerate neutrophils, 5% lymphocytes, and 53% macrophages. B, Immunocytochemistry stain on the same specimen for coronavirus antigen showing positive staining
in association with macrophages.

may be used (Table 20-5). When antigen tests are positive,
provided the test is performed and interpreted properly (with
use of positive and negative control slides), studies suggest
that only cats with FIP have positive test results.44,55 False-
negative results occur when there are insufficient numbers of
infected cells, when low quantities of virus are present, or
when antigen is unavailable for detection because of complex-
ing by antibody.
Pathologic Findings
Gross Pathologic Findings
At necropsy, gross findings in cats with FIP include variable
quantities of pleural, pericardial, and peritoneal effusion (Figure
20-10, A). Fibrin adhesions may be present and the mesentery
may be clumped. Abdominal organs may be enlarged or irreg-
ular. Granulomas appear as variably sized multifocal white,
cream, tan, or yellow nodular lesions on serosal surfaces and
within the parenchyma of organs such as the lungs, spleen, kid-
neys, pancreas, and liver (see Figure 20-10, B). Lesions have also
been described within the nasal cavity and sinuses. Pyogranulo-
mas may be visible grossly as miliary lesions, or they may be
several centimeters in diameter. Thoracic and/or abdominal
lymphadenomegaly is a common finding. Diffuse or focal thick-
ening of the intestinal wall or intestinal mass lesions may be
present. Examination of the brain can reveal fibrinous exudate
in association with the meninges, with or without ventricular
dilation and hydrocephalus (see Figure 20-4). Thymic involu-
tion may also be present.
 CHAPTER 20  Feline Coronavirus Infection 203

TABLE 20-5
Assays Available for Diagnosis of Feline Infectious Peritonitis
Assay Specimen Type Target Performance
Fluorescent or Wash or effusion FCoV Gold standard for diagnosis. False negatives can occur in
immunoperoxidase specimens, tissue specimens that contain low numbers of macrophages or virus
antibody staining aspirates, tissues particles, or when virus is complexed by antibody. Immuno-
obtained at biopsy fluorescence is more sensitive than immunoperoxidase methods.
or necropsy Non-specific staining may be interpreted as positive results by
untrained personnel.
Serology Blood, CSF, aqueous FCoV antibody Positive antibody titers reflect only antibodies to a coronavirus
humor, effusion and are not specific for a diagnosis of FIP. Most cats in multicat
fluid households test positive. Negative titers can occur in cats with
advanced FIP. High titers in cats that do not reside in multicat
households and that have signs suggestive of FIP may support
the diagnosis. Interlaboratory variation in methodology and
titer reporting occurs.
RT-PCR Blood, wash or FCoV RNA Does not differentiate between virulent and avirulent FCoV
­effusion specimens, strains, and avirulent strains may be found in tissues and blood.
tissue aspirates, Sensitivity and specificity can vary depending on assay design.
tissues obtained at False negative results occur when virus levels are low, when
biopsy or necropsy variant virus strains are present, or as a result of degradation of
viral nucleic acid during specimen transport.
Histopathology Usually necropsy Inflammatory Biopsy is often not feasible antemortem as a result of critical ill-
specimens, but also ­lesions induced ness and coagulopathies.
biopsies by FCoV (pyo-
granulomatous
vasculitis)

FCoV, feline coronavirus; FIP, feline infectious peritonitis; RT-PCR, reverse transcriptase–polymerase chain reaction.

Histopathologic Findings
The characteristic histopathologic findings of FIP are systemic
perivascular, multifocal to coalescing pyogranulomatous or
granulomatous inflammatory lesions (Figure 20-11). Lesions
predominantly contain macrophages and neutrophils, with
lesser numbers of lymphocytes and plasma cells, although occa-
sionally the histiocytic or the lymphoplasmacytic component
of the inflammatory response is more florid. Necrosis may be
present within the lesions. Lesions in the central nervous sys-
tem consist of pyogranulomatous meningoencephalomyelitis
and choroiditis. Other findings that may be identified include
lymphoid depletion, which results from apoptosis, and mem-
branous glomerulonephritis.
Treatment and Prognosis
Currently, no cure for FIP exists; it is a progressive, invari-
ably fatal disease. The goal of treatment is to prolong life span
and improve quality of life through reduction of inflamma-
tion and supportive care. The most effective treatment known
is prednisolone, administration of which results in temporary
remissions in some cats (Table 20-6). Other immunosuppres-
sive drugs, such as chlorambucil and cyclophosphamide, have
been used in addition to prednisolone, but whether these drugs
improve outcome is unknown, and they have the potential to be
toxic. A variety of immunomodulators and antiviral drugs have
been tried, such as ribavirin and oral and parenteral human
recombinant IFN-α, but none have convincingly shown ben-
efit in vivo. Prolonged remissions were reported in several cats
treated with a combination of glucocorticoids and feline IFN-
ω,56 but a randomized, placebo-controlled clinical trial reported
no effect of feline IFN-ω.57 Other drugs used to treat cats with
FIP include the immunomodulatory drugs pentoxifylline and
Polyprenyl Immunostimulant (see Chapter 7)58; ozagrel hydro-
chloride, a thromboxane synthetase inhibitor59; and the antivi-
ral drug nelfinavir.14,60 Controlled clinical trials are required to
assess the efficacy and safety of these treatments. Because FIP is
an immune-mediated disease, nonspecific immune stimulation
has the potential to cause harm. The use of small interfering
RNA molecules, which bind viral RNA and prevent viral repli-
cation, has recently shown promising results in vitro.61 Cyclo-
sporin inhibits FIPV replication in vitro;62 studies are required
to determine if cyclosporin treatment benefits infected cats or
whether harm results from immunosuppression.
Supportive treatments that may be required include subcu-
taneous fluid therapy and nutritional support. Inappetent cats
can benefit from enteral nutrition through a feeding tube. The
use of broad-spectrum antimicrobial drugs to treat cats with FIP
is controversial; it may only promote opportunistic infections
with resistant bacteria.
The prognosis for cats with FIP is generally grave. Almost all
cats with effusion at the time of diagnosis die within weeks. Very
rarely, more prolonged survival times (1 to 2 years) have been
documented after glucocorticoid treatment (see Case Example).
204 SECTION 1  Viral Diseases

B
FIGURE 20-10  A, Gross necropsy findings in a 10-month-old male intact domes-
tic shorthair cat with effusive FIP. Approximately 1.2 liters of yellow fluid was present in
the abdomen, and there are abundant fibrin strains adherent to the visceral and parietal
peritoneal surfaces. B, Kidneys of the cat in Figure 20-8. Multiple, pale tan firm nodules
expand the renal cortices and protrude from the cortical surfaces. (Courtesy of the Univer-
sity of California, Davis Veterinary Anatomic Pathology service, D. Gasper and M. Jones.) B
FIGURE 20-11  A, Histopathology of the liver of a cat with FIP. There is pyogranulo-
matous hepatitis. Hematoxylin and eosin stain. B, Macrophages stain strongly positive for
The median survival time in one study of 37 cats was 9 days feline coronavirus antigen with immunohistochemistry. (Courtesy Dr. Patricia Pesavento,
University of California, Davis Veterinary Anatomic Pathology service.)
(range, 3 to 200 days).57 In another it was 21 days (range, 1
to 99 days) for 30 cats with effusive disease, 38 days (range,
1 to 171 days) for 12 cats with noneffusive disease, and 111
days (range, 7 to 477 days) for 9 cats with mixed effusive and TABLE 20-6
noneffusive disease.14 Hyperbilirubinemia, the presence of effu-
sion, and lymphopenia are negative prognostic factors20; in one Suggested Drug Dosages for Treatment of Feline
study, the hematocrit, lymphocyte count, and serum albumin, Infectious Peritonitis
potassium, sodium, and globulin concentrations decreased as
disease progressed, and total bilirubin concentration and serum Interval
liver enzyme activities increased.14 Euthanasia should be consid- Drug Dose (mg/kg) Route (hours)
ered for cats with severe illness that fail to respond to treatment
Prednisolone 1-2 PO 12-24
within a 3-day period.20
Chlorambucil* 2 mg/cat PO 48-72
Immunity and Vaccination *Monitor the CBC during treatment.

Although antibodies to the spike protein can neutralize virus,

antibodies are required for FIP to occur, because FIP is an
immune complex disease. Cell-mediated immunity is important
for protection,6 but if immunity is incomplete, granulomatous
or pyogranulomatous disease results.
The development of vaccines for FIP has been complicated
by the fact that stimulation of antibody production against
FCoV can accelerate the disease, should FIP develop after vacci-
nation has been performed. Antibodies may bind to Fc receptors
on macrophages and accelerate virus uptake in a phenomenon
known as antibody-dependent disease enhancement (ADDE).
Currently, an intranasal, temperature-sensitive mutant serotype
II FIP virus vaccine is available on the market, but its use has
been controversial. The vaccine virus replicates in the lower
temperatures found in the respiratory tract. It is licensed for
administration from 16 weeks of age, by which time most kit-
tens have already been exposed to FCoV. The vaccine does not
appear to cause ADDE,63-65 but its efficacy and ability to induce

immunity against heterologous strains is controversial. In a
study of 138 cats that belonged to 15 different cat breeders,
virtually all of which were seropositive, there was no difference
in prevalence of FIP in vaccinated versus placebo-treated cats.63
A slight reduction in the prevalence of FIP occurred when the
vaccine was used in cats that had not been exposed to FCoV
before vaccination, but protection was not convincing based on
the small numbers of cats that developed the disease in each
group.6,65
Prevention
In households that contain only one or a few cats, young cats
that develop FIP likely become infected with FCoV before they
are acquired. They may or may not have FIP at the time of
acquisition. When a cat from a single-cat household dies with
FIP, it is suggested that the owner wait at least 2 months before
a new cat is obtained, so that any virus in the environment
becomes inactivated.20 Selection of a new cat from a different
genetic background than the previous cat should be considered,
and if possible, the breeder should be informed if a purebred
cat develops FIP. If a low number of other cats remain in the
household, they may or may not continue to shed virus. These
cats often have a positive antibody titer, but this in no way
predicts that they will develop FIP. Before a new cat is intro-
duced to a household that has a history of FIP, factors that
could reduce stress and overcrowding should be identified and
addressed.
The risk of transmission and disease can be reduced through
attention to hygiene, prevention of overcrowding, maintenance
of a larger ratio of adult to juvenile cats, and ensuring that cats
are in stable groups of three or fewer per room. Cats should
have sufficient numbers of regularly cleaned litter trays located
in a different area from where they are fed. Methods to control
FIP in cattery situations, such as identification and removal of
CASE EXAMPLE
Signalment: “Ricky”, a 9-month-old male castrated domestic
shorthair from Sacramento, CA
History: Ricky was brought to a local veterinary clinic because
of increased thirst and urination. A serum chemistry panel
showed hyperglobulinemia (7.7 mg/dL), and urinalysis
showed a specific gravity (SGr) of 1.025 with an inactive
sediment; aerobic bacterial urine culture was negative. Three
days later, Ricky became inappetent and was returned to the
local veterinary clinic. Laboratory abnormalities included
mature neutrophilia (11,904 cells/µL), lymphocytosis (5104
cells/µL), eosinophilia (1536 cells/µL), hyperglobulinemia
(6.9 mg/dL), and hypoalbuminemia (2.4 mg/dL). A feline
coronavirus antibody titer was 1:400. Serology for Toxoplasma
gondii was negative. Plain thoracic radiographs showed
a mild interstitial pattern. Abdominal ultrasound showed
mesenteric lymphadenomegaly, and an aspirate of the
lymph nodes showed lymphoid reactivity. Treatment with
cyproheptadine was initiated, and Ricky’s appetite recovered,
CHAPTER 20  Feline Coronavirus Infection 205
chronic shedders with serial fecal RT-PCR assays and removal
of kittens from the queen followed by isolation at 5 to 6 weeks
of age (before maternal antibody has declined), have limita-
tions and are difficult to achieve properly in large catteries.20
For example, cats that do not shed FCoV may still be infected
with avirulent FCoV strains, and shedding may recommence at
a later date.22 Isolation of kittens may be useful if reexposure
is prevented until after they are 16 weeks of age, when their
immune system is more mature.6 In shelter situations, FIP may
be reduced when overcrowding and prolonged stays are mini-
mized, especially during kitten season.6 If possible, owners
that adopt cats from shelter environments should be provided
with a handout that provides basic information on the disease
(and other major infectious diseases of shelter cats such as ret-
rovirus infections, bartonellosis, and feline upper respiratory
tract disease) and the ubiquitous nature of infection.
Further understanding of genetic factors that contribute to
FIP is required, because selective breeding may reduce the risk
of the disease. In the meantime, the breeding of cats that pro-
duce litters that succumb to FIP should be avoided. This is espe-
cially true for male cats, because a single male cat can have an
effect on far more kittens and litters than a single queen. It is
recommended that no more than six breeding animals be main-
tained if possible.6
Public Health Aspects
There is no evidence that humans can become infected with
FCoV. The closest human coronavirus relative is the severe
acute respiratory syndrome (SARS) coronavirus. Other corona-
viruses cause FIP-like disease in nonfelids such as ferrets and
mice. If a coronavirus emerged that could cause similar clinical
manifestations and outcomes in humans as FIPV can in cats, it
would represent a major threat to humans and would be the
subject of intense research.
after which treatment was discontinued. For the 3 weeks
that followed, the cat had been appetent and energetic, but
occasional soft feces had been noticed in the litter box. The
owners were concerned about the possibility of FIP.
Ricky was obtained at 3 months of age from a rescue group,
who rescued him as an 8-week-old stray kitten. As a kitten
he had multiple upper respiratory tract infections, but since
adoption he had been healthy and shared a household with
one other cat. He was an indoor cat that was sometimes
walked briefly outdoors. He was fed commercial dry and wet
cat food.
Physical Examination:
Body Weight: 4.2 kg
General: Bright, alert and responsive, hydrated. T = 103°F
(39.4°C), HR = 200 beats/min, eupneic.
All Systems: No clinically significant abnormalities of any body
system were detected. Body condition score was 5/9.
Laboratory Findings:
CBC:
HCT 27.3% (30%-50%)
MCV 42.1 fL (42-53 fL)
206 SECTION 1  Viral Diseases
MCHC 34.1 g/dL (30-33.5 g/dL)
WBC 24,950 cells/µL (4500-14,000 cells/µL)
Neutrophils 17,141 cells/µL (2000-9000 cells/µL)
Lymphocytes 6886 cells/µL (1000-7000 cells/µL)
Monocytes 724 cells/µL (50-600 cells/µL)
Eosinophils 200 cells/µL (150-1100 cells/µL)
Basophils 25 cells/µL (0-50 cells/µL)
Platelets 518,000/µL (180,000-500,000 platelets/µL).
Serum Chemistry Profile:
Sodium 147 mmol/L (151-158 mmol/L)
Potassium 4.5 mmol/L (3.6-4.9 mmol/L)
Chloride 116 mmol/L (117-126 mmol/L)
Bicarbonate 18 mmol/L (15-21 mmol/L)
Phosphorus 6.4 mg/dL (3.2-6.3 mg/dL)
Calcium 9.5 mg/dL (9.0-10.9 mg/dl)
BUN 22 mg/dL (18-33 mg/dL)
Creatinine 1.1 mg/dL (1.1-2.2 mg/dL)
Glucose 83 mg/dL (63-118 mg/dL)
Total protein 10.7 g/dL (6.6-8.4 g/dL)
Albumin 2.6 g/dL (2.2-4.6 g/dL)
Globulin 8.1 g/dL (2.8-5.4 g/dL)
ALT 31 U/L (27-101 U/L)
AST 17 U/L (17-58 U/L)
ALP 35 U/L (14-71 U/L)
Gamma GT <3 U/L (0-4 U/L)
Cholesterol 143 mg/dL (89-258 mg/dL)
Total bilirubin < 0.1 mg/dL (0-0.2 mg/dL).
Serum Protein Electrophoresis: A polyclonal gammopathy
with a mild decrease in albumin concentration and mild
increases in the α2 and β1 fractions was present (see Figure
20-4). These changes were consistent with the acute-phase
inflammatory response.
Imaging Findings: Abdominal ultrasound: The spleen
was moderately enlarged. There was diffuse mesenteric
lymphadenopathy (see Figure 20-8, B).
Mesenteric Lymph Node Aspirate Cytology: Intact nucleated
cells were composed of a heterogenous population of
lymphocytes, predominated by small, mature lymphocytes.
Lower numbers of intermediate and large reactive
lymphocytes, moderate numbers of mildly degenerate
neutrophils, and scattered plasma cells and histiocytes
were noted. Immunocytochemistry using two different
monoclonal antibodies against FCoV was negative, but
macrophages were low in number.
Microbiologic Testing: FeLV antigen and FIV antibody
serology: negative
Serology (IFA) and blood culture for Bartonella clarridgeiae and
Bartonella henselae: negative
Serology (IFA) for vector-borne diseases: negative for antibod-
ies to Ehrlichia canis, Neorickettsia risticii, Anaplasma spp.,
and Rickettsia spp.
PCR for FCoV (whole blood): negative
PCR panel for other bloodborne pathogens (Anaplasma phago-
cytophilum, Anaplasma platys, Bartonella spp., E. canis, N. ris-
ticii, Mycoplasma haemofelis): negative
Aerobic and anaerobic bacterial culture of mesenteric lymph
node aspirate: negative
Serology for FCoV: positive at 1:102,400
Diagnosis: A tentative diagnosis of FIP was made on the basis
of Ricky’s background, the marked polyclonal gammopathy,
and the strongly positive coronavirus titer.
Treatment and Outcome: Biopsy of the enlarged mesenteric
node was offered, but the owners declined. Ricky was treated
with prednisolone (5 mg PO q12h for 7 days, followed by 5
mg PO q24h thereafter), chlorambucil (2 mg PO every 3 days),
and pentoxifylline (50 mg PO q8h). Six weeks later, the cat
was well and CBC variables within reference ranges. Serum
total protein concentration was 8.5 g/dL, with a globulin
concentration of 4.5 g/dL. Abdominal ultrasound examination
showed persistent but mild mesenteric lymphadenomegaly
(0.5 to 0.75 cm in diameter). A feline coronavirus titer was
1:25,600. Treatment with feline interferon-ω was commenced
(4.5 million units SC once weekly). Ricky was seen again 3
months later, at which time he continued to be playful and
appetent, with a stable body weight of 4.5 kg. A CBC showed
mild anemia (HCT 29%), a neutrophil count of 4439 cells/µL,
and lymphopenia (468 cells/µL). A chemistry panel and
abdominal ultrasound showed no abnormalities. Chlorambucil
and interferon-ω were discontinued. The next time the cat was
reexamined was 12 months after the onset of illness, at which
time he continued to be apparently healthy. CBC findings
were unchanged, and the serum globulin concentration
was 4.7 g/dL. The prednisolone dose was decreased to 5 mg
q48h and pentoxifylline treatment was discontinued. One
month later, albumin and globulin concentrations were 3.3
and 5.1 g/dL, respectively, and the prednisolone dose was
reduced to 2.5 mg q48h. At the next 1-month recheck, a CBC
was unremarkable but globulin was 5.7 g/dL. Abdominal
ultrasound showed mildly enlarged mesenteric lymph nodes,
and the serum coronavirus antibody titer was 1:409,600. The
prednisolone dose was increased to 5 mg PO q24h; 1 month
later, the serum globulin concentration was 4.9 g/dL. One and
a half years after the onset of illness, Ricky was still apparently
healthy according to the owner, but a midabdominal mass was
palpated on physical examination, and the serum globulin
concentration had increased again (5.6 g/dL). Abdominal
ultrasound showed several moderately enlarged and
hypoechoic lymph nodes in the ileocolic region, the largest
of which was 0.9 cm in diameter. The surrounding mesentery
was focally hyperechoic. There was also focal hyperechoic
retroperitoneal tissue surrounding the right kidney with scant
retroperitoneal fluid. Attempts to obtain aspirates from the
lymph nodes were unsuccessful. The prednisolone dose was
increased to 5mg PO q12h, and treatment with chlorambucil
and pentoxifylline was reinstituted.
One week later, Ricky developed lethargy and inappetence. A
CBC showed macrocytic anemia (HCT 24.3%, MCV 57.2 fL)
and lymphopenia (782 cells/µL). A serum chemistry panel
showed only hyperglobulinemia (5.8 g/dL). FeLV and FIV
serology was repeated and was again negative, and the
coronavirus antibody titer was 1:25,600. Treatment with
cyproheptadine was initiated and the chlorambucil and
pentoxifylline discontinued. However, inappetence contin-
ued, and persistent pyrexia (103.4° to 104°F), hematoche-
zia, and tachypnea developed over the next few days. The
hematocrit dropped to 16.5%, and hypokalemia, hyponatre-
mia, and hypochloremia were identified. Ricky was hospital-
ized and treated with 1 unit of packed RBC, IV crystalloids,
and parenteral antimicrobial drugs. Thoracic radiographs
showed a severe, diffuse, patchy alveolar and nodular inter-
stitial pattern with thickening of the bronchial walls and
mild pleural effusion (see Figure 20-7). A tracheobronchial

lavage showed marked mixed, predominantly pyogranulo-
matous inflammation with moderate epithelial hyperplasia
and some degenerate neutrophils (see Figure 20-9). Immu-
nocytochemistry with an anti-FCoV antibody was strongly
positive in macrophages. Aerobic and anaerobic bacterial
cultures of the wash specimen were negative. The cat subse-
quently seizured and was euthanized.
Necropsy Findings: Necropsy showed moderate to severe,
multifocal to coalescing pyogranulomatous capsulitis and
serositis that involved the spleen, liver, kidney, intestines,
diaphragm, thoracic and abdominal walls, and pericardium.
There was also multifocal pyogranulomatous splenitis,
hepatitis, nephritis, meningoencephalitis, and pneumonia
with necrosis. Straw-colored effusion was present in
the abdominal cavity, thoracic cavity, and pleural space.
Immunohistochemistry was strongly positive for FCoV
antigen (see Figure 20-11).
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Pedersen NC. A review of feline infectious peritonitis virus infection:
1963-2008. J Feline Med Surg. 2009;11:225-258.
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2012;41:558-567.
51. Boettcher IC, Steinberg T, Matiasek K, et al. Use of anti-corona-
virus antibody testing of cerebrospinal fluid for diagnosis of feline
infectious peritonitis involving the central nervous system in cats.
J Am Vet Med Assoc. 2007;230:199-205.
52. Foley JE, Lapointe JM, Koblik P, et  al. Diagnostic features of
clinical neurologic feline infectious peritonitis. J Vet Intern Med.
1998;12:415-423.
53. Lewis KM, O’Brien RT. Abdominal ultrasonographic findings
associated with feline infectious peritonitis: a retrospective review
of 16 cases. J Am Anim Hosp Assoc. 2010;46:152-160.
54. Pratelli A. Comparison of serologic techniques for the detec-
tion of antibodies against feline coronaviruses. J Vet Diagn Invest.
2008;20:45-50.
55. Tammer R, Evensen O, Lutz H, et  al. Immunohistological dem-
onstration of feline infectious peritonitis virus antigen in paraf-
fin-embedded tissues using feline ascites or murine monoclonal
antibodies. Vet Immunol Immunopathol. 1995;49:177-182.
56. Ishida T, Shibanai A, Tanaka S, et  al. Use of recombinant feline
interferon and glucocorticoid in the treatment of feline infectious peri-
tonitis. J Feline Med Surg. 2004;6:107-109.
57. Ritz S, Egberink H, Hartmann K. Effect of feline interferon-omega
on the survival time and quality of life of cats with feline infectious
peritonitis. J Vet Intern Med. 2007;21:1193-1197.
58. Legendre AM, Bartges JW. Effect of Polyprenyl Immunostimulant
on the survival times of three cats with the dry form of feline infectious
peritonitis. J Feline Med Surg. 2009;11:624-626.
59. Watari T, Kaneshima T, Tsujimoto H, et al. Effect of thromboxane
synthetase inhibitor on feline infectious peritonitis in cats. J Vet Med
Sci. 1998;60:657-659.
60. Hsieh LE, Lin CN, Su BL, et  al. Synergistic antiviral effect of
Galanthus nivalis agglutinin and nelfinavir against feline coronavirus.
Antiviral Res. 2010;88:25-30.
61. McDonagh P, Sheehy PA, Norris JM. In vitro inhibition of feline
coronavirus replication by small interfering RNAs. Vet Microbiol.
2011;150:220-229.
62. Tanaka Y, Sato Y, Osawa S, et al. Suppression of feline coronavirus
replication in vitro by cyclosporin A. Vet Res. 2012;41-43.
63. Fehr D, Holznagel E, Bolla S, et al. Placebo-controlled evaluation of a
modified live virus vaccine against feline infectious peritonitis: safety
and efficacy under field conditions. Vaccine. 1997;15:1101-1109.
64. Postorino Reeves NC, Pollock RV, Thurber ET. Long-term follow-
up study of cats vaccinated with a temperature-sensitive feline infectious
peritonitis vaccine. Cornell Vet. 1992:82.
65. Postorino Reeves NC. Vaccination against naturally-occurring FIP
in a single large cat shelter. Feline Pract. 1995;23:81-82.
CHAPTER 21
Feline Immunodeficiency Virus Infection
Jane E. Sykes
Overview of Feline Immunodeficiency Virus
Infection
First Described: California, 1986 (Pedersen)1; serologic evi-
dence of infection dates back to the 1960s2,3
Cause: Feline immunodeficiency virus (FIV) (family Retroviri-
dae, subfamily Orthoretrovirinae, genus Lentivirus)
Affected Hosts: Domestic and wild cats; hyenas
Geographic Distribution: Worldwide
Mode of Transmission: Biting, and to a lesser extent transpla-
cental, through milk, possibly venereal, blood transfusion
Major Clinical Signs: Lethargy, fever, pallor, stomatitis, diar-
rhea, muscle atrophy, neurologic signs, signs of under-
lying neoplastic or immune-mediated disorders or
opportunistic infections
Differential Diagnoses: FeLV infection, bartonellosis, other
causes of stomatitis such as feline calicivirus infection,
immune-mediated disease, other chronic inflammatory
and neoplastic diseases of cats that occur in the absence
of detectable infection with FIV.
Human Health Significance: FIV does not infect humans.
Etiology and Epidemiology
Feline immunodeficiency virus (FIV) is an enveloped, RNA
virus that belongs to the Lentivirus genus of the Retroviridae.
It infects domestic and wild cats worldwide, as well as hyenas.4
Like HIV, FIV establishes a chronic, persistent infection that, in
some cats, ultimately culminates in immunodeficiency. Because
of its similarities to HIV, FIV infection in cats has been used as
a research model for HIV infection and acquired immunodefi-
ciency syndrome (AIDS),5 and relative to other viral infections
of companion animals, much is known about its pathogenesis.
Knowledge of retroviral structure and replication is required
in order to understand diagnostic, treatment, and prevention
strategies that target these viruses. Like other retroviruses, FIV
has a three-layered structure that is composed of an inner-
most genome-nucleocapsid complex with helical symmetry, an
icosahedral capsid, and an envelope with glycoprotein spikes
(­Figure 21-1). The FIV genome contains three major genes: gag,
which encodes the virion core proteins (capsid [p24], nucleo-
capsid, and matrix); pol, which encodes the reverse transcrip-
tase, protease and integrase enzymes; and env, which encodes
surface (gp120) and transmembrane virion (gp41) envelope
glycoproteins. Several other accessory and regulatory genes are
also present. FIV invades cells via the primary receptor CD134,
which is expressed on feline CD4+ T cells, B cells, and activated
macrophages,6,7 and the secondary receptor CXCR4, which
is normally a chemokine receptor. The viral envelope fuses
with the cell membrane, and the capsid enters the cytoplasm,
where reverse transcription occurs and a double-stranded DNA
(dsDNA) copy of the retroviral genome is made. Additional
sequences, known as long terminal repeats (LTRs), are added
to either end of the viral genome. The virus then passes into the
nucleus where the dsDNA copy integrates into the host genome;
at this point the integrated dsDNA becomes a provirus. Tran-
scription of this DNA, which is controlled by the LTRs, leads
to synthesis of new virion components, and virus assembly and
budding occur at the cell surface (Figure 21-2). Depending on
the cellular environment, proviral DNA may either be latent,
whereby transcription does not occur, or be transcriptionally
active. Latency is one mechanism by which retroviruses can
evade the host immune system. Because the reverse transcrip-
tase enzyme is prone to error, the mutation rate of retroviruses
is high, and a diversity of viral variants continuously emerges
from infected hosts. Integration of retroviral DNA into host cell
DNA can disrupt genes that are responsible for cell growth and
differentiation (proto-oncogenes). Alternatively, cellular onco-
genes captured and carried by retroviruses develop mutations
during retroviral replication, and are then re-inserted into host
cell DNA. The end result is abnormal cell growth and differen-
tiation, which results in tumor formation by some retroviruses.
Based on sequence diversity of the env gene, there are six
different subtypes of FIV, A through F. Subtypes A and B are
distributed most widely, followed by subtype C8-10—although a
recent study from the United States showed an equal distribu-
tion of subtypes A, B, and C (Table 21-1).11-31 In addition, a
number of recombinant subtypes have been recognized, such
as A/B, A/C, B/D, B/E, and A/B/C recombinants, and additional
subtypes also likely exist.9,11 The heterogeneity of the virus
complicates the design of molecular diagnostic tests and vac-
cines for FIV. Whether differences in the clinical manifestations
of disease relate to infection by different subtypes requires fur-
ther study.9,10,31
Retroviruses survive only minutes outside the host and
are very susceptible to disinfection. FIV is shed in high con-
centrations in saliva, and the major mode of transmission is
through bites. Transplacental transmission, transmission dur-
ing parturition, and through milk have been documented
experimentally, but these modes appear to be uncommon in
naturally infected cats, and kittens infected by this route may
not sustain productive infection.32,33 Venereal transmission has
not been demonstrated, but FIV can be recovered from semen,
and experimental inoculation of FIV into the vaginas of queens
results in transmission.34,35 Transmission also has the potential
to occur through blood donation.
209
210 SECTION 1  Viral Diseases
p17
matrix
RNA
CD134
T lymphocyte
surface
FIGURE 21-1  Structure of FIV. An FIV virion is shown adjacent to the surface of a CD4+ T cell. Lentiviruses contain two identical strands of RNA (the viral genome) and associated
enzymes, which include reverse transcriptase, integrase, and protease, packaged into a core composed of the p24 capsid protein with a surrounding p17 protein matrix, all enclosed by a
phospholipid membrane envelope that is derived from the host cell. Viral encoded membrane proteins (gp41 and gp120) are bound to the envelope. CD134 and CXCR4 (chemokine) recep-
tors on the host cell surface function as the receptors for FIV.
Seropositivity to FIV (which is equivalent to infection because
of viral persistence) is consistently associated with a history of
bite wounds, older age, male sex, illness, and outdoor access
(Table 21-2).12,36-41 Being of mixed breed has also been a risk
factor in some studies. The mean age at diagnosis is around 6
to 8 years, and 80% to 90% of cats are more than 2 years of
age. Male cats are up to 4.7 times more likely to be seroposi-
tive than female cats.40 Indoor housing decreases transmission
but does not eliminate it.42 Worldwide, the seroprevalence of
FIV in domestic pet cats ranges from around 1% to 12%.9,40,41
A study that included more than 18,000 North American cats
estimated the overall prevalence of FeLV and FIV infections as
around 2.3% and 2.5%, respectively.5 Higher prevalences are
found in feral and free-ranging cats and sick cats. In the North
American study, the prevalence of FIV infection in sick, feral
cats was 18.2%, whereas that in healthy indoor cats was only
0.7%. A study from France showed a seroprevalence of 21% in
unowned cats, compared with 10% in owned cats.43 In Japan,
the overall seroprevalence is very high (23% in one study), and
as many as one-third of male cats test positive.12 Occasionally,
co-infections with FeLV occur, and infection with one retrovirus
is a risk factor for infection with the other.36,37
Clinical Features
Signs and Their Pathogenesis
The main cellular target for FIV is the CD4+ T cell. How-
ever, FIV also infects CD8+ T cells, B cells, macrophages, and
Lipid bilayer
gp41
gp120
Reverse
transcriptase
Protease
Integrase
p24 capsid
Chemokine
receptor
dendritic cells, microglia, and astrocytes. The subsequent effects
of the virus on the immune system are complex, incompletely
understood, and seem to result in both immune suppression and
immune activation.
Three phases of disease have been delineated, acute (pri-
mary), subclinical, and terminal, although not all phases are
recognized in many naturally infected cats. After inoculation,
the virus replicates in lymphoid tissues, and high concentrations
of virus are present in blood 2 weeks after infection. A peak
of viremia occurs 8 to 12 weeks after infection (Figure 21-3).
There is a decline in CD4+ and CD8+ T cells in peripheral
blood. This may be associated with transient illness, which lasts
3 to 6 months and is often unrecognized by cat owners. Some
cats show lethargy, fever, anorexia, diarrhea, stomatitis, weight
loss, and/or lymphadenopathy during the acute phase. Lymph-
adenopathy results from lymphoid hyperplasia, and can persist
for weeks to months. Neutropenia can also occur,44 possibly
as a result of neutrophil apoptosis. CD4+/CD25+ T regulator
(Treg) cells are infected and activated during the acute phase.
These cells then inhibit the proliferation of activated CD4+ and
CD8+ T cells, and cause them to undergo apoptosis. This may
contribute to persistence of FIV and further immunosuppres-
sion.45-47 Altered dendritic cell function may also occur.48,49 In
general, impaired T cell function in acute infection is thought to
result from cytokine dysregulation, immunologic anergy (failure
to respond to specific antigens), and increased apoptosis.45 Nev-
ertheless, most cats survive this phase because of a rebound in
CD8+ T cell numbers and a strong humoral immune response.
 CHAPTER 21  Feline Immunodeficiency Virus Infection 211

Virion binding
to CD134 and
chemokine
receptor New FIV
Fusion of FIV virion
membrane with host
FIV virion cell membrane; entry
of viral genome
into cytoplasm
Plasma
membrane Cytokine

Cytokine
receptor FIV
gp120/ Budding
Chemokine gp41
CD134 and release of
receptor
mature virion
FIV RNA
genome

Reverse
transcriptase–
mediated synthesis
of proviral DNA
Cytokine activation
of cell; transcription
Integration of of FIV genome;
provirus into transport of viral FIV core
host cell RNAs to cytoplasm structure
genome

Synthesis of
FIV proteins;
FIV DNA assembly of virion
provirus core structure
FIV RNA
transcript
Nucleus

FIGURE 21-2  The life cycle of FIV. The sequential steps in FIV reproduction are shown, from initial infection of a host cell to release of a new virus particle. An infected cell produces
many virions, each capable of infecting nearby cells, with subsequent spread of the infection.

hyperactivation. Some studies also describe a sustained increase

TABLE 21-1
Distribution of FIV Subtypes Worldwide
A Australia, New Zealand, United States (especially
western United States), South Africa, northwestern
Europe, Japan, United Kingdom11-19
B Central and eastern United States, Caribbean, central
and western Europe, Brazil, eastern Japan11,14,20-25
C United States, Canada, New Zealand, southeast
Asia11,12,26-29
D Japan, Vietnam, rarely United States21,27,31
E Argentina10,30
F United States10,31
In the subclinical (or asymptomatic) phase, CD4+ T cell
numbers rebound, and the plasma virus load declines to very
low levels. Cats remain subclinically infected, often for years
or even for life. This is not latent infection, because virus pro-
duction continues at low levels; a slow, progressive decline in
CD4+ T cell numbers; reduction in the CD4+:CD8+ T cell ratio;
and in some cats, hyperglobulinemia, which results from B cell
in CD8+ T cell numbers. Although activated, paradoxically, T
cells have a reduced ability to respond to antigenic stimulation.
Altered lymphocyte expression of cell surface molecules (includ-
ing CD4 and cytokine receptors, and MHC II antigens), and
continued alteration of dendritic cell and neutrophil function
also contribute to immunosuppression. Dysregulation of cyto-
kine production occurs. For example, cats that are chronically
infected with FIV fail to produce Il-2, Il-6, and Il-12 in response
to Toxoplasma gondii infection and instead produce elevated
levels of the antiinflammatory cytokine Il-10.45,50 There is a
slow influx of immune cells into the brain, with gradual pro-
gression of central nervous system (CNS) disease.51 The rate of
progression of the subclinical phase depends on factors such as
the virus strain, co-infections with other agents that activate
virus transcription, and host immunity.
In some cats, these changes ultimately lead to the terminal
phase, which is characterized by clinical signs of opportunistic
infections, neoplastic disease, myelosuppression, and neurologic
disease. This is the phase most commonly recognized in natu-
rally infected cats (Table 21-3). However, many infected cats
never develop FIV-related clinical signs, even when CD4+ T cell
counts are low, and instead die from other causes. The terminal
phase of FIV infection is commonly associated with moderate
to severe periodontal disease, lymphoplasmacytic stomatitis
(Figure 21-4), gingivitis, and feline odontoclastic resorptive
212 SECTION 1  Viral Diseases

lesions,52 which may result from opportunistic bacterial and

TABLE 21-2 viral infections. Other opportunistic infections include chronic
Risk Factors for FIV Infection in Cats Seen at the UC Davis VMTH bacterial skin and ear infections, persistent viral upper respira-
tory tract infections, dermatophytosis, mycobacterial infections,
Number Number fungal infections such as cryptococcosis and sporotrichosis,
of FIV+ of Control Odds hemoplasmosis, toxoplasmosis, and/or parasitic infections such
Variable Cats* Cats† Ratio P-value‡ as demodecosis and severe flea burdens. With the exception of
stomatitis, the relationship between many of these opportunistic
Sex infections and FIV infection is somewhat unclear, because the
Male neutered 100 117 — prevalence of many of these infections in cats with FIV infection
Male intact 10 5 2.729 0.08 is similar to that in cats without FIV infection. However, infec-
Female/female 17 88 0.24Åò <0.001 tions are often more severe and less responsive to treatment than
neutered the same infections in immunocompetent cats.
Breed The development of neoplasia in FIV-infected cats is thought
Mixed 116 170 — to result primarily from immune suppression, although there is
Purebred 11 40 0.37 0.02 serologic evidence that cats may be infected with a gammaher-
Environment pesvirus similar to Epstein-Barr virus that might be reactivated
Indoor 15 57 — in cats infected with FIV.53 Lymphomas are the most commonly
Outdoor 85 107 2.58 0.01 reported FIV-associated tumor, especially B cell lymphomas, but
also T cell and non-B, non-T cell lymphomas.54 FIV-infected
Stray history
cats are 5 times more likely to develop lymphoma than cats not
No 101 195 —
infected with FIV and are more likely to develop it an earlier age.
Yes 26 15 3.73 <0.001
Leukemia and squamous cell carcinomas (SCCs) are also com-
Cats in household mon tumors in cats with FIV, although the association with SCC
<3 34 70 — may be confounded by outdoor exposure. Other tumors include
≥3 39 61 1.25 0.53 mast cell tumors, fibrosarcomas, meningiomas, and metastatic
carcinomas; some infected cats develop more than one type of
Modified from Trott KA, Kass PH, Sparger EE, et  al. A clinical case
neoplasia. Rarely, lymphoma may develop as a result of viral inte-
control study: clinical presentation of FIV-positive cats. University of
California, Davis, STARS in Science Day. 2007; abstr. gration into the genome and disruption of proto-oncogenes.54,55
*All cats were >6 months of age and none had a history of FIV vaccination. Immune dysregulation and increased circulating immune
†All control cats were negative for FIV antibody at their visit to the complexes in the terminal phase can lead to immune-mediated
University of California, Davis. disorders, such as immune-mediated glomerulonephritis and
‡P-values <0.05 were significant. uveitis.56 Myelodysplasia develops in some cats,57,58 which
ÅòIn other words, female cats were four times less likely to be FIV+ than
may be manifested by clinical signs of lethargy, inappetence,
male neutered cats. pallor, or evidence of bleeding tendencies such as petechial
hemorrhages.

1200
-Possible acute HIV syndrome Death
-Wide dissemination of virus
1000 -Seeding of lymphoid organs
Opportunistic
infections 1:256
800
Plasma viremia titer
CD4+ T cells/mm3

Clinical latency

600 Constitutional 1:64

symptoms
400 1:16

200 1:4

0 0
0 6 12 1 5 10
Weeks Years
FIGURE 21-3  Changes in virus load and CD4+ T Cell numbers over the course of infection with HIV. A similar clinical course of infection occurs for FIV infection. Viremia is detected early
after infection and may be accompanied by systemic signs (acute phase). Plasma viremia then falls to very low levels and remains this way for many years (subclinical phase). Although this period
is referred to here as “clinical latency,” the virus itself is not latent during this time, because there is ongoing production of virus and a steady decline in CD4+ T cell counts. In the terminal phase
of infection, signs of immunodeficiency develop and plasma viremia increases. Some infected cats never reach this phase. Antibody production declines, and antibody tests may be negative in
cats with advanced terminal-phase disease, but PCR assays are more likely to be positive.
 CHAPTER 21  Feline Immunodeficiency Virus Infection 213

TABLE 21-3
Disease Diagnoses in 127 Cats with FIV Infection and 210 Age-
Matched Control Cats Seen over the Same Time Period at the UC
Davis VMTH*
Cats Infected
with FIV Controls
Disease (n = 127)† (n = 210)‡ P-value§
Intraocular 16 17 0.18
­inflammation
Stomatitis 21 11 < 0.001
Cardiomyopathy 21 17 0.02
Upper respiratory 13 9 0.03 A
tract disease
Neurologic signs 16 24 0.75
Chronic kidney 23 40 0.83
disease
Diabetes mellitus 9 18 0.63
Hyperthyroidism 11 14 0.49
Lymphoma 23 20 0.02
Squamous cell 12 9 0.057
­carcinoma (SCC)
Fibrosarcoma 2 9 0.17
Carcinomas other 4 12 0.28
than SCC

Modified from Trott KA, Kass PH, Sparger EE, et  al. A clinical case
control study: clinical presentation of FIV-positive cats. University of
California, Davis, STARS in Science Day. 2007; abstr.
*Results should be interpreted with caution because the data were retro-
spectively collected. Some cats may have had undiagnosed disease, such
as subclinical chronic kidney or cardiac disease. The pathogenesis of a
disease process in FIV-positive cats might also differ from that found in B
FIV-negative cats.
†All cats were >6 months of age and none had a history of FIV vaccina- FIGURE 21-4  severe lymphoplasmacytic stomatitis in a cat infected with feline
tion.
immunodeficiency virus. A, There is marked hyperemia, ulceration, and proliferative
‡All control cats were negative for FIV antibody at their visit to the lesions in the palatoglossal folds. (From Bonello D, Roy CG, Verstraete FJM. Non-neoplastic
University of California, Davis.
proliferative oral lesions. In: Verstraete FJM, Lommer MJ, eds. Oral and Maxillofacial Sur-
§As determined by chi-square (univariate) analysis. P-values <0.05 were gery in Dogs and Cats. Philadelphia, PA: Saunders; 2011.) B, Histopathology of a biopsy
significant.
from a cat with severe stomatitis associated with FIV infection.

degenerative changes in other organs, such as the myocardium

The end result of infection by neurovirulent strains of FIV
can be progressive behavioral changes such as increased aggres-
sion and cognitive disturbances, tremors, sleep disturbances,
anisocoria, delayed reflexes, abnormal cranial nerve function,
urinary and fecal incontinence, and seizures. Decreased nerve
conduction velocities and abnormal electroencephalograms and
brainstem evoked potentials have been documented.59 The virus
does not infect neurons, but neuronal cell death occurs through
multiple incompletely defined mechanisms.51
Inflammatory lesions can develop in a variety of organs in
cats infected with FIV. An inflammatory myopathy has been
described,60 and terminally, severe muscle atrophy can occur.
Infection is associated with gut inflammation and intestinal epi-
thelial cell damage (also known as AIDS enteropathy), which may
result in chronic diarrhea and failure to gain weight.61 The extent
to which chronic FIV infection contributes to inflammatory or
and the kidneys, is not well understood, because the prevalence
of cardiomyopathy and interstitial nephritis in geriatric cats not
infected with FIV is high (see Table 21-3).
When transplacental transmission occurs in FIV-infected
queens, only some kittens in a litter may be infected. In the first
year of infection, the overall rate of such transmission is around
70%.62 Rates of mother-to-kitten transmission are highest in
queens that have a CD4+ count less than 200 cells/µL, those
with signs of immunodeficiency, and those infected within the
past 15 months.63 In utero transmission can lead to arrested
fetal development, abortion, stillbirth, low birth weights, and
the birth of T cell deficient kittens.62,64
Physical Examination Findings
Many cats infected with FIV have no signs of illness, or clini-
cal signs that are unrelated to FIV infection. Cats in the acute
214 SECTION 1  Viral Diseases
TABLE 21-4
Diagnostic Assays Available for Feline Immunodeficiency Virus Infection
Assay Specimen Type Target
Serology (ELISA, Blood, serum Antibody to FIV
Western blotting,
IFA)
PCR Blood FIV proviral DNA
or viral RNA
Virus ­isolation Blood FIV
IFA, immunofluorescent antibody.
phase of FIV infection may be febrile or show generalized
peripheral lymphadenopathy. Behavioral abnormalities such
as obtundation, aggression, and cognitive impairment may be
evident in cats with terminal neurologic disease; other neuro-
logic abnormalities include ataxia, anisocoria, and abnormal
segmental reflexes. Ocular abnormalities may be present as
a result of the FIV infection itself, opportunistic infections,
or lymphoma, and include anterior uveitis, hyphema, pars
planitis, chorioretinitis, and/or glaucoma.65 Other common
physical examination findings in cats with terminal disease are
periodontal disease and chronic stomatitis, signs of chronic
upper respiratory tract infection, otitis externa, pyoderma,
cutaneous abscesses, and a diverse and variable spectrum of
disease manifestations that relate to other underlying opportu-
nistic infections or neoplasia.
Diagnosis
For many cats, infection with FIV is diagnosed during screening
efforts. Screening for infection should be performed with tests
that detect antibody against FIV, because these assays have the
highest overall sensitivity and are rapid and widely available
(Table 21-4). It has been recommended that the retrovirus status
of all cats be known regardless of the presence of absence of ill-
ness.66 Indications for testing as recommended by the American
Association of Feline Practitioners (AAFP) are shown in Box
21-1. In practice, compliance with retrovirus testing is low. In
a study that evaluated 967 cats with bite wounds or cutaneous
abscesses that presented to veterinary practitioners from 134
practices in 30 states, the combined FeLV-FIV status of only 96
(9.9%) of the cats was known.38 In addition, despite the avail-
ability of a financial incentive for retesting, only 64 of 478 cat
owners returned their cats for retesting after treatment.
When positive test results occur in sick cats, the role that FIV
plays as a cause of the signs may be unclear, although it is reason-
able to assume that FIV may be playing a role in cats with severe
stomatitis, unusual infections with intracellular pathogens such
as mycobacteria, and lymphoma. With refinement and improved
Performance
Positive test results in cats >6 months of age that have not been
vaccinated for FIV equal infection, but confirmation is recom-
mended in healthy cats using a second test from a different
manufacturer or Western blot. False positives occur in cats <6
months of age (maternal antibody) and cats vaccinated with the
FIV vaccine. False negatives can occur in the first 2 months of in-
fection or with advanced disease and severe immunosuppression.
Sensitivity and specificity vary between laboratories. Even well-
designed assays may be insensitive because of viral variability
and low-level viremia. PCR assays are the only assay that can
definitively diagnose infection in cats vaccinated for FIV. Never
use without performing serology.
Difficult, not widely available. Used primarily as a research tool.
BOX 21-1
Indications for Serologic Testing for FIV and FeLV
• Sick cats, even if tested negative in the past
• All newly acquired cats or kittens (2 tests, at least 60
days apart)
• After exposure to a retrovirus-infected cat or a cat
with unknown status, and especially after a bite wound
(2 tests, at least 60 days apart)
• Cats that live in a household with other retrovirus-
infected cats (annual retesting unless isolated)
• Before initial vaccination with FeLV or FIV vaccines
• Before use as a blood donor (in conjunction with real-
time PCR)
• On entry to a shelter or before adoption (2 tests, at
least 60 days apart); if financial resources are not avail-
able for this, cats should be held singly and postadop-
tion testing (2 tests, at least 60 days apart) recommend-
ed before mingling with other cats occurs. The status of
the other cats in the household should be known before
new cats are introduced.
• For group-housed cats, before introduction (2 tests, at
least 60 days apart if possible) and on an annual basis
• Testing is considered optional for feral cat trap-neuter-
return programs
Modified from Levy J, Crawford C, Hartmann K, et al. 2008 Ameri-
can Association of Feline Practitioners’ feline retrovirus management
guidelines. J Feline Med Surg 2008;10:300-316.
availability of test methodologies in the future, clinical assess-
ment of CD4+ T cell counts, the CD4+/CD8+ ratio, and plasma
viral loads may facilitate interpretation of the relationship
between disease manifestations and infection and provide prog-
nostic information, as in human patients infected with HIV.67
 CHAPTER 21  Feline Immunodeficiency Virus Infection
Laboratory Abnormalities
Complete Blood Count
Common abnormalities on the CBC in cats infected with FIV
include mild anemia, lymphopenia, and neutropenia. Occasion-
ally severe anemia, thrombocytopenia, thrombocytosis, mono-
cytopenia, or leukocytosis occur. In one large European study,
neutrophil counts of FIV-infected cats were lower than those of
control cats, and lymphocyte counts were higher than those of
control cats.68 Leukopenia and neutropenia were more likely to
be present in FIV-infected cats.
Serum Biochemical Tests
The most common and significant abnormality on the chemis-
try panel in cats infected with FIV is hyperproteinemia, which
results from increased γ-globulin concentrations and is a direct
result of FIV infection (rather than the result of opportunistic
infection). Total protein concentrations that ranged from 4.5 to
11 g/dL were reported in one study.68 Other findings are vari-
able and relate to the presence of concurrent disease, neoplasia,
or opportunistic infections.
Urinalysis
Urinalysis may reveal proteinuria in cats with glomerulonephritis.
Coagulation Testing
Cats infected with FIV can have mild prolongations in their
APTT, thrombin time, and fibrinogen concentrations, although
the PT and platelet function are generally normal.69
Cytologic Evaluation of Bone Marrow
Bone marrow cytologic evaluation in FIV-infected cats with
cytopenias and nonregenerative anemias may show mild dys-
plasia (usually not as severe as in FeLV infections); erythroid
hypoplasia; and/or myeloid hyperplasia despite peripheral leu-
kopenia, sometimes with a left shift. The latter suggests ineffec-
tive hematopoiesis or maturation arrest.
Microbiologic Tests
Serologic Diagnosis
The initial assay of choice for diagnosis of, and screening for,
FIV infection is an ELISA assay that detects antibody to FIV.
Provided there has not been a history of vaccination for FIV
and the tested cat is less than 6 months of age, positive anti-
body test results equate with infection, because the virus estab-
lishes a lifelong, persistent infection. Point-of-care, lateral-flow
ELISA assays and diagnostic laboratory-based ELISA assays
are in widespread use and have rapid turnaround times and
high sensitivities and specificities.70 These assays usually detect
antibodies to the FIV p24 core protein, and sometimes to the
gp40 transmembrane protein. False positive test results occur
rarely as a result of operator error or nonspecific reactivity
against tissue culture components after vaccination.71 When
Western immunoblotting was used as the gold standard, the
sensitivities, specificities, and positive and negative predic-
tive values of six different ELISA assays (Synbiotics Witness,
Viracheck FIV, IDEXX SNAP Combo Plus, PetChek Plus
Anti-FIV, MegaCor Fastest, and Bio Veto Duo Speed) in one
U.S. study of 535 cats with an overall FIV seroprevalence of
10.3% ranged from 94.5% to 100%, 98.5% to 100%, 91.2%
to 100%, and 99.2% to 100%, respectively.70 The Mapic FIV
test had a high (23.1%) rate of invalid test results and was
not recommended for clinical practice. When these assays are
215
used to screen healthy cats for infection, confirmation of posi-
tive results is recommended because of the low prevalence of
infection in this population of cats and the higher possibility
that false-positive test results may occur. Confirmation can be
done using an assay from a different manufacturer or Western
blotting. However, Western blotting is technically demanding,
subject to interoperator variability in interpretation, and it may
be less sensitive than ELISA assays when performed by some
laboratories.72
Positive ELISA assay results in the absence of FIV infec-
tion can occur in cats that have been vaccinated for FIV, or
kittens less than 6 months of age that possess maternal anti-
body (because of infection or vaccination of the queen). No
currently available serologic test, including Western immu-
noblotting, distinguishes between natural infection and vacci-
nation or the presence of maternal antibody. Kittens that test
positive should be retested after 6 months of age (the Advisory
Bureau on Cat Diseases recommends retesting after 4 months
of age).66,73 Nevertheless, kittens less than 6 months of age
should still be tested, because the vast majority of negative
kittens will be declared free of infection. Molecular testing
could be considered to confirm infection in kittens that test
positive at less than 6 months of age (see later discussion).
Cats with a history of FIV vaccination may remain antibody-
positive for more than 4 years.66 Because infection can occur
in the face of vaccination, positive test results in a vaccinated
cat may represent infection and/or historical vaccination.
Currently, molecular testing with PCR assays is required to
identify infection in these cats, but some infected cats test
PCR-negative. An ELISA assay has been developed that can
distinguish naturally infected from vaccinated cats, but this
assay is not commercially available. The assay was used to
test blood samples from 73 uninfected, unvaccinated cats; 89
uninfected, FIV-vaccinated cats; and 102 FIV-infected cats,
including 3 cats that had been vaccinated.9 The assay had a
sensitivity of 97% and a specificity of 100% for detection of
FIV infection in these cats.
False-negative ELISA assay results occur early in the course
of illness, because cats may take up to 60 days to develop an
antibody response.66 Rarely, antibody production is delayed
for 6 months or longer. Thus when recent exposure is possible,
testing should be repeated a minimum of 2 months later. False-
negative test results also occur in cats in the terminal phase
of disease, as a result of impaired antibody production, or in
kittens with rapidly progressive infections. These cats often
have high plasma viral loads. Thus, if advanced FIV infection
is suspected, negative test results should be followed by virus
detection using PCR. When serology is used as a screening test,
negative results are considered to be highly reliable because of
the high sensitivity of the test and the low prevalence of infec-
tion in most populations of healthy cats.66
Immunofluorescent antibody (IFA) assays that detect anti-
bodies to FIV have also been described. When performed by
experience laboratory personnel, these have sensitivities and
specificities that range from 95% to 100% when compared with
Western immunoblotting.71
Molecular Diagnosis Using the Polymerase Chain Reaction
A variety of PCR assays have been developed for diagnosis of
FIV infection. Assays may detect viral RNA (reverse transcrip-
tase [RT]-PCR), proviral DNA, or both RNA and proviral DNA
in peripheral blood. Because the FIV vaccine is inactivated and
216 SECTION 1  Viral Diseases
A B
FIGURE 21-5  A, Depletion of paracortical areas in the mesenteric lymph node of an 8-year-old female spayed domestic shorthair with FIV infection that was euthanized as a result of
the development of progressive neurologic signs. B, Normal feline lymph node for comparison purposes.
does not replicate or integrate into the host genome, PCR assays
should not detect vaccine virus in cats that have been vacci-
nated for FIV. Compared with serology, PCR can be insensitive
(sensitivity <80%), because viral loads in healthy cats are often
extremely low, and some strains may not be detected because
of variability in the sequence of the viral genome among FIV
isolates. Sensitivity is likely to be higher in cats in the acute
and terminal phases of disease when viral loads are higher, but
this requires further study. False-positive test results have the
potential to occur as a result of laboratory contamination; in
some commercial laboratories, unacceptable sensitivities and
specificities have been reported (as low as 41% and 44%,
respectively).74,75 In advertising materials, IDEXX Laborato-
ries report an assay sensitivity and specificity of 80.5% and
99.9%, respectively, among 36 FIV-infected cats, 96 uninfected
vaccinated cats and 92 uninfected unvaccinated cats, with no
difference in specificity in the latter two groups.76 A FRET real-
time PCR assay directed at the gag gene has been described
that differentiates between FIV subtypes (see Chapter 5 for an
explanation of FRET PCR assays).11 The assay was positive in
60% of 101 cats with positive antibody tests for FIV. A history
of vaccination with the FIV vaccine was present in 13 of the
cats, and all these cats tested negative. The vaccination history
of the remaining cats was unknown. Given their limitations,
PCR assays should not be used for diagnosis of FIV infection
in the absence of concurrent serologic testing, and the results
of PCR assays should not be used to decide whether vaccina-
tion should be performed. Cats that are seronegative that test
positive with PCR may be in the terminal phase of disease and
unable to produce antibody, or the PCR assay result may be a
false positive. Latently infected cats have rarely been described
that test positive for proviral DNA but negative for antibody.76
These cats lack any immunologic or clinical abnormalities that
occur with active infection.76 Whether these cats are likely to
develop productive infection (as reported in humans with latent
HIV infection) is unknown.77
Virus Isolation
FIV can be isolated from peripheral blood lymphocytes in pri-
mary feline T cell cultures, but this is technically demanding and
expensive and can take 2 to 3 weeks, so it is not used for routine
diagnostic purposes.
Pathologic Findings
Gross pathologic findings in cats with FIV infection include
emaciation, stomatitis, lymphadenopathy, and evidence of
secondary neoplasia or opportunistic infections. Evidence of
disorders of aged cats, such as hyperthyroidism, cardiomy-
opathy, and interstitial nephritis, may also be present. Histo-
pathologic changes in cats with FIV infection include follicular
hyperplasia or, in the chronic phase, lymphoid depletion in
the paracortical regions and plasma cell infiltration of lymph
nodes (Figure 21-5); lymphoplasmacytic ulcerative stomatitis
(see Figure 21-4, B); and bone marrow pathology as described
previously in this chapter. Evidence of chronic interstitial
nephritis, sometimes with membranous glomerulonephritis
and glomerulosclerosis, may be present. Inflammatory changes
may be seen within skeletal muscle and/or the gastrointesti-
nal tract. Histopathology of the CNS in cats infected with
neurovirulent FIV strains may show mild lymphoplasmacytic
meningitis, perivascular lymphocytic infiltrates, diffuse gliosis,
microglial nodules, and mild neuronal degeneration and apop-
tosis.59,78 Neuronal dysfunction may be more important than
neuronal loss.59
Treatment and Prognosis
Supportive Care
Cats in the terminal phase of FIV infection may require fluid
therapy, nutritional support, regular dental prophylaxis, dilute
chlorhexidine-based mouth washes or oral gels, and dental
extractions and antimicrobial drugs with activity against anaer-
obes for severe stomatitis (Table 21-5). Whole mouth extrac-
tions have been beneficial for some cats with severe stomatitis,
but it is critical that all tooth roots be removed. Referral to a
board-certified veterinary dentist should be considered. Topi-
cal glucocorticoids (e.g., 1% prednisolone acetate, every 4 to
12 hours depending on severity) and topical atropine are indi-
cated for cats with anterior uveitis. Systemic glucocorticoids
should only be used if absolutely necessary, because their use
 CHAPTER 21  Feline Immunodeficiency Virus Infection
TABLE 21-5
Suggested Medications for Treatment of Cats in the Terminal Phase of Feline Immunodeficiency Virus Infection
Drug Dose Route Interval (hours) Comments
Zidovudine (AZT) 5 to 15 mg/kg PO 12
Human recombinant 1 to 50 U/cat PO 24
interferon alpha
0.12% Chlorhexidine 1 mL Topical 12 to 48
plus zinc gluconate
oral gel
Clavulanic acid­– 12.5 to 25 mg/kg PO 12
amoxicillin
Lactoferrin 200 mg powder or Topical 24
40 mg/kg solution
Prednisolone 5 mg PO 24
is associated with increased plasma viremia. Some cats with
advanced neurologic signs show clinical improvement after
treatment with glucocorticoids. Opportunistic infections may
respond to appropriate antimicrobial treatment, but prolonged
or lifelong treatment may be required. Griseofulvin should not
be used to treat dermatophytosis, because it has been associated
with bone marrow suppression in FIV-positive cats.79 Recom-
binant human erythropoietin or darbepoetin may be useful for
some cats with nonregenerative anemia and does not appear
to increase viral load through activation of transcription of
latent virus.80 Although recombinant human granulocyte col-
ony stimulating factor (G-CSF) can increase neutrophil counts
in cats infected with FIV, antibodies can develop within a few
weeks that cross-react with endogenous G-CSF. When recombi-
nant human granulocyte-macrophage colony stimulating factor
(GM-CSF) was used, an increase in virus load occurred, so the
use of these drugs is not recommended.
Antiviral and Immunomodulator Drugs
Topical lactoferrin administration has been associated with
clinical improvement in some FIV-infected cats with stomatitis
(see Chapter 7). Cats with stomatitis or neurologic disease may
benefit from oral or subcutaneous treatment with zidovudine
(AZT), but AZT can cause bone marrow suppression, so the
CBC must be monitored weekly for the first month and monthly
thereafter. Treatment is not recommended for cats with myelo-
suppressive disease.73 AZT should be discontinued if the hema-
tocrit drops below 20%, after which anemia usually resolves in
a few days.73 AZT-resistant FIV mutants can develop as early as
6 months after the start of treatment. Fozivudine (45 mg/kg PO
q12h) reduces viremia in cats with acute FIV infection and may
be less likely to produce hematologic adverse effects.81 Plerixa-
for (AMD3100), a selective antagonist of the CXCR4 recep-
tor, is active against FIV in vitro.82 When used in 40 naturally
infected cats for 6 weeks in a placebo-controlled double-blind
217
May have benefit for cats with neurologic signs
or stomatitis. Monitor CBC (see text). Toxicity
may be more likely at doses above 5 mg/kg.
For stomatitis
For stomatitis
For stomatitis
For refractory gingivitis and stomatitis. Must be
purchased from chemical suppliers.
For refractory stomatitis or advanced neurologic
disease. May also be required for lymphoma or
other CNS neoplasms. Use only if absolutely
necessary.
study, a decreased proviral load was reported without evidence
of adverse effects, but there was no improvement in clinical or
immunologic variables.73,83
Results of treatment with human recombinant IFN-α have
been mixed, but prolonged survival was reported in 24 FIV-
infected cats after oral administration of a low dose of the drug
when compared with 6 untreated control cats.83 Clinical scores
and laboratory parameters improved in some sick, naturally
infected cats that were treated with recombinant feline IFN-ω,
despite no significant changes in viral load.84
Management of FIV-Infected Cats
Cats that test positive for FIV should be housed indoors to
prevent transmission to other cats, as well as to protect them
from other infections. The latter is important even in subclini-
cally infected cats, as other infections have the potential to acti-
vate viral transcription and accelerate disease progression. The
feeding of raw foods and hunting behavior should be avoided.
Cats should be neutered to reduce the chance of roaming and
aggressive interactions with other cats. Minimally, FIV-posi-
tive cats should be rechecked every 6 months in order to moni-
tor body weight, assess for periodontal disease, and discuss the
need for routine laboratory testing and vaccination with core
vaccines. A complete physical examination, CBC, serum bio-
chemistry panel, and urinalysis are recommended on an annual
basis.66 At least during acute FIV infection, cats can mount
an immune response to vaccines,85,86 but because vaccination
with core vaccines may activate viral transcription, vaccination
should be performed only for FIV-positive cats that are likely
to be exposed to other cats. The use of inactivated vaccines
is recommended, because of the potential for vaccine-induced
disease in immunosuppressed cats.73 When hospitalized, FIV-
infected cats should be kept in separate cages away from
isolation, where there may be other cats with transmissible
infectious diseases. Perioperative antimicrobial drug treatment
218 SECTION 1  Viral Diseases
could be considered for cats that undergo invasive surgery or
dental treatments.66,73
Prognosis
A limited number of studies have shown no significant difference
in life span between FIV-infected cats and uninfected cats.42,87
In one study, the median survival times of these two groups
after testing for FIV infection were 3.9 (39 cats) and 5.9 (22
cats) years, respectively.87 In a larger study of more than 1000
FIV-infected cats and more than 8000 age- and sex-matched
control cats, the median survival times of the two groups were
4.9 years and 6.0 years, respectively.66 The progression and
severity of disease is related to virus strain and host immunity.
Infection of geriatric and neonatal cats is associated with more
rapid progression and severity of disease than infection of young
adult cats.84,88 For example, progression to terminal immuno-
deficiency can occur within 2 months in kittens.89 Because the
life span of FIV-infected cats may not differ greatly from that
of uninfected cats, no cat should be euthanized on the basis of
a positive FIV test alone.66,73 However, other factors, such as
control of the infection in group-housed or breeding cats, may
necessitate euthanasia or rehoming of some cats that test posi-
tive. Once terminal FIV-related disease occurs, life spans are
typically less than 1 year.
Immunity and Vaccination
The FIV vaccine first became available in the United States in
July 2002 (Fel-O-Vax FIV, Fort Dodge Animal Health) and is
an inactivated, adjuvanted, whole-virus vaccine that contains
FIV Petaluma (subtype A) and FIV Shizuoka (subtype D). The
vaccine has been available in New Zealand and Australia since
2004. It is licensed for the vaccination of healthy cats that are 8
weeks of age or older as an aid in the prevention of FIV, and in
experimental challenge studies, protected 60% to 80% of cats
from infection. The introduction of the vaccine generated con-
troversy, because (1) existing serologic assays cannot differenti-
ate between natural infection and vaccination, (2) vaccination
provides only partial protection from infection, and (3) PCR
assay results cannot be relied upon in vaccinated cats. Although
concerns exist that the vaccine may only protect against infec-
tion with strains from homologous subtypes, studies have shown
moderate protection against infection with subtype B strains.90,91
In one study, 10 of 14 cats were protected from subtype B infec-
tion when challenged 1 year after vaccination compared with
none of 5 control cats.90 However, 13-week-old vaccinated cats
were not protected from challenge with FIV subtype A strain
Glasgow-8; all vaccinated and control cats were infected.92
Because of diagnostic test interference, uncertain efficacy in
the field, and the increased risk of sarcoma formation associ-
ated with adjuvanted vaccines, the FIV vaccine is considered a
noncore vaccine that should only be used for cats at high risk
of infection, such as outdoor cats that fight with other cats. The
initial vaccine should only be given to cats that test negative
with FIV ELISA assays. Cat owners should be informed that
protection is incomplete and that identification of subsequent
infection may be impossible in some cats with the available diag-
nostic tests. The initial vaccine dose is followed by two booster
doses at 3- to 4-week intervals, followed by annual boosters
so long as risk persists. An identification microchip should be
placed that links FIV vaccination to the microchip number, so
should the cat escape or be relinquished to a shelter, a positive
antibody test result is not interpreted as FIV infection.
Prevention
Transmission of FIV is reduced when cats are housed indoors.
When one positive cat is identified in a household, all other
cats should be tested and no new cats should be introduced,
as this may lead to conflict and increased fighting behavior.
Isolation of positive cats in a household should be considered.
In breeding catteries, infection can be prevented by screening
all new introductions, preferably with a 2-month quarantine
period for introductions followed by retesting, and removal
of all infected queens and toms from the cattery. Transmis-
sion in shelters can be reduced by testing on intake or housing
cats singly in cages, and testing at or shortly after adoption,
together with education of adopters in regard to the need to
retest for infection.
Surgical needles, endotracheal tubes, breathing circuits, and
instruments should never be shared between cats without proper
cleaning and disinfection, and all cats used as blood donors
should test negative for FIV antibodies with ELISA and, if pos-
sible, PCR assays offered by a diagnostic laboratory that has
a high level of quality control. Fluid lines and multidose vials
should not be shared between cats, because they can become
contaminated with body fluids.
Public Health Aspects
Despite its similarities to HIV, no evidence of natural infection
of humans with FIV exists.
 CHAPTER 21  Feline Immunodeficiency Virus Infection
CASE EXAMPLE
Signalment: “Arthur” an 11-year-old male neutered domestic
shorthair from northern California
History: Arthur was initially brought to his veterinarian for a
2-day history of lethargy and decreased appetite. Physical
examination was unremarkable. Blood work showed a
hematocrit of 28% (reference range, 29% to 38%), a white
cell count of 6400 cells/µL with a normal differential,
clumped platelets, mild hyperbilirubinemia (0.5 mg/dL),
mild hypoalbuminemia (2.4 g/dL), and hyperglobulinemia
(5.5 g/dL). He was treated with amoxicillin and mirtazapine,
but lethargy and inappetence persisted. Serial hemograms
performed over the subsequent 3 months showed
progressive leukopenia due to a neutropenia and
lymphopenia (Table 21-6), during which time Arthur was
treated with orbifloxacin, with some improvement in his
appetite and attitude. A bone marrow aspirate performed 5
weeks after the onset of illness showed myeloid hyperplasia
with a left shift. Serology for FIV and FeLV was performed
11 weeks after onset of illness, and the cat was negative
for FeLV antigen and had an equivocal test result for FIV
antibody. Serum thyroxine concentration was low (0.7 mcg/
dL, reference range 1.1 to 3.3 mcg/dL). The cat was referred
for further evaluation.
Arthur was an indoor and outdoor cat and had a history of pre-
dation (mice and birds) and fighting with other cats in the
neighborhood. His diet otherwise consisted of a commercial
wet cat food. He was vaccinated regularly for rabies, FeLV,
feline herpesvirus-1, feline calicivirus, and feline panleuko-
penia virus infections.
Current Medications: Orbifloxacin, 7 mg/kg PO q12h
Other Medical History: Routine blood work had been
performed 3 years previously, which showed a normal CBC,
a globulin of 5 g/dL, a urine specific gravity of 1.069, and a T4
of 1.3. At that time, Arthur tested negative for FeLV antigen
and positive for FIV antibody.
Physical Examination:
Body Weight: 3.1 kg
General: Quiet, alert, responsive, estimated to be 5% to 7%
dehydrated, T = 103.9° F (39.9° C), HR = 144 beats/min,
RR = 50 breaths/min, mucous membranes pale pink, CRT
< 2 s. A dry haircoat with moderate amounts of scale was
present. There was no evidence of ectoparasites.
Eyes, Ears, Nose, and Throat (with Dilated Fundoscopic
Examination): Moderate gingivitis and absent mandibular
canine teeth. No other clinically significant abnormalities
were detected.
Musculoskeletal: Body condition score 2/9. Generalized
muscle atrophy was noted. Ambulatory in all four limbs.
Cardiovascular: Aside from an intermittent gallop rhythm, no
abnormalities were noted.
Respiratory: No abnormalities were detected.
Gastrointestinal and Urogenital: Abdomen soft and
nonpainful on palpation, mild hepatomegaly. Urinary
bladder was small and soft.
Lymph Nodes: All lymph nodes less than 1 cm in diameter.
219
Laboratory Findings:
CBC:
HCT 22.5% (30%-50%)
MCV 47.5 fL (42-53 fL)
MCHC 32.9 g/dL (30-33.5 g/dL)
Reticulocytes 7400 cells/µL
WBC 1100 cells/µL (4500-14,000 cells/µL)
Neutrophils 704 cells/µL (2000-9000 cells/µL)
Band neutrophils 99 cells/µL
Lymphocytes 220 cells/µL (1000-7000 cells/µL)
Monocytes 77 cells/µL (50-600 cells/µL)
32,000 platelets/µL (180,000-500,000 platelets/µL).
Neutrophils showed moderate toxicity with many Döhle
bodies, and there were many macroplatelets.
Serum Chemistry Profile:
Sodium 149 mmol/L (151-158 mmol/L)
Potassium 3.1 mmol/L (3.6-4.9 mmol/L)
Chloride 115 mmol/L (117-126 mmol/L)
Bicarbonate 17 mmol/L (15-21 mmol/L)
Phosphorus 4.3 mg/dL (3.2-6.3 mg/dL)
Calcium 8.8 mg/dL (9.0-10.9 mg/dl)
BUN 17 mg/dL (18-33 mg/dL)
Creatinine 1.1 mg/dL (1.1-2.2 mg/dL)
Glucose 158 mg/dL (63-118 mg/dL)
Total protein 8.1 g/dL (6.6-8.4 g/dL)
Albumin 3.0 g/dL (2.2-4.6 g/dL)
Globulin 5.1 g/dL (2.8-5.4 g/dL)
ALT 121 U/L (27-101 U/L)
AST 113 U/L (17-58 U/L)
ALP 10 U/L (14-71 U/L)
Creatine kinase 278 U/L (73-260 U/L)
Gamma GT <3 U/L (0-4 U/L)
Cholesterol 204 mg/dL (89-258 mg/dL)
Total bilirubin 0.8 mg/dL (0-0.2 mg/dL)
Magnesium 2.4 mg/dL (1.5-2.5 mg/dL).
Anti-nuclear Antibody Serology: Positive at 1:32
Microbiologic and Virologic Testing: Point-of-care ELISA
serology for FeLV antigen and FIV antibody: negative
Feline coronavirus antibody serology: negative at 1:25
Aerobic and anaerobic blood cultures (three specimens): negative
PCR for FIV proviral DNA: positive (cycle threshold value = 30,
values <40 are positive)
Imaging Findings:
Abdominal Ultrasound: Hepatomegaly was identified and
the spleen had a mottled echotexture. There was mild
enlargement of the ileocecal lymph nodes and mild diffuse
thickening of the small intestinal submucosal layer.
Thoracic Radiographs: Cardiopulmonary structures were
within normal limits.
Cytologic Findings:
Liver Aspiration Cytology: A moderately bloody background
contained scattered, variably sized clusters of uniform
hepatocytes and low numbers of nucleated cells. The nucleated
cells consisted of a mixture of nondegenerate neutrophils,
lymphocytes, macrophages, and rare myeloid precursors.
Several macrophages were erythrophagic. Interpretation:
Possible mild myeloid extramedullary hematopoiesis.
Spleen Aspiration Cytology: The specimen was highly cellular
with a pink background that contained a moderate amount
220 SECTION 1  Viral Diseases
of blood and scattered clumps of hemosiderin. A mixed
population of lymphocytes was admixed with variable
numbers of hematopoietic precursor cells. Hematopoietic
precursors were primarily granulocytic precursors, with
many progranulocytes. A few erythroid precursors, including
erythroblasts, were noted. Plasma cells were moderately
increased in number, and both immature and mature forms
were seen. Several binucleated and rare trinucleated plasma
cells and scattered immature lymphocytes were noted.
Vacuolated macrophages were increased in number and
often contained phagocytized erythrocytes or blue-green
pigment consistent with hemosiderin. Scattered mitotic
figures were noted. Interpretation: Moderate plasmacytosis,
extramedullary hematopoiesis, and histiocytic hyperplasia
with erythrophagocytosis and increased iron. The large
number of immature plasma cells raised the possibility of
plasma cell neoplasia; however, reactive plasmacytosis could
not be ruled out.
Bone Marrow Aspiration Cytology: Numerous hypercellular
unit particles were present. A few mature megakaryocytes
were noted. Hematopoietic cells consisted largely of myeloid
precursors, with an estimated myeloid to erythroid ratio
greater than 10. Myeloid cells showed orderly maturation to
the band neutrophil stage, with few segmented neutrophils.
Few erythroid precursors were noted, which included rare
prorubricytes, rubricytes, and metarubricytes. Scattered well-
differentiated plasma cells and a small amount of hemosiderin
were noted. Interpretation: Marked myeloid hyperplasia,
TABLE 21-6
Progression of Laboratory Abnormalities in an 11-Year-old Male Neutered Domestic Shorthair That Tested Positive
for Antibodies to FIV 3 Years Earlier, at Which Time a CBC was Normal
Days after Onset of Illness
39 47 56
Hematocrit (%) 29 29 33
White blood cells/µL 2000 2100 1000
Neutrophils/µL 1180 1407 610
Lymphocytes/µL 700 588 340
Platelets/µL 191,000 196,000 Clumped
FIV/FeLV serology*
*SNAP FIV/FeLV Combo Test, IDEXX Laboratories.
SUGGESTED READINGS
Goldkamp CE, Levy JK, Edinboro CH, et  al. Seroprevalences of feline
leukemia virus and feline immunodeficiency virus in cats with abscesses
or bite wounds and rate of veterinarian compliance with current guide-
lines for retrovirus testing. J Am Vet Med Assoc. 2008;232:1152-1158.
Hartmann K. Clinical aspects of feline immunodeficiency and feline leuke-
mia virus infection. Vet Immunol Immunopathol. 2011;143(3-4):190-
201 (for a review of molecular pathogenetic mechanisms of disease).
Hosie MJ, Addie D, Belak S, et  al. Feline immunodeficiency. ABCD
guidelines on prevention and management. J Feline Med Surg.
2009;11:575-584.
marked erythroid hypoplasia, and moderate megakaryocytic
hypoplasia. The myeloid hyperplasia together with persistent
neutropenia suggested myelodysplasia.
Serum Protein Electrophoresis: A mild increase in α2
globulins was present (1.34 g/dL, reference range, 0.4-0.9
g/dL) together with a broad-based gamma region. This was
consistent with an acute-phase inflammatory response.
Diagnosis: Terminal phase of FIV infection, characterized by
pancytopenia secondary to myelodysplasia; hepatopathy
(open diagnosis).
Comments: The progression of FIV infection was apparent
in this cat, which transitioned from a seropositive to a
seronegative, PCR-positive state as a result of progressive
decline in immune function and, ultimately, failure to
produce antibodies. The cause of the hepatopathy was
not identified. Blood cultures were negative, and spleen
and liver aspiration cytology failed to reveal evidence of
underlying neoplasia. Because of the plasmacytosis in the
spleen, serum protein electrophoresis was performed to
assist diagnosis of a plasma cell tumor (as supported by a
monoclonal gammopathy), but this was not apparent. The
cat was treated with clavulanic acid–amoxicillin without
clinical improvement. Treatment with glucocorticoids was
also offered because of the possibility of undiagnosed
round cell neoplasia or immune-mediated neutropenia (in
light of the myeloid hyperplasia combined with a positive
antinuclear antibody test). The cat died at home 1 month
later, and necropsy was not performed.
74 83 Reference Range
27 22 30-50
1100 1100 4500-14,000
572 704 2000-9000
462 220 1000-7000
Clumped 32,000 180,000-500,000
Equivocal/ Negative/
negative negative
Levy J, Crawford C, Hartmann K, et al. 2008 American Association of
Feline Practitioners’ feline retrovirus management guidelines. J Feline
Med Surg. 2008;10:300-316.
REFERENCES
1. Pedersen NC, Ho EW, Brown ML, et al. Isolation of a T-lympho-
tropic virus from domestic cats with an immunodeficiency-like syn-
drome. Science. 1987;235:790-793.
2. Gruffydd-Jones TJ, Hopper CD, Harbour DA, et  al. Serological
evidence of feline immunodeficiency virus infection in UK cats from
1975-76. Vet Rec. 1988;123:569-570.
 CHAPTER 21  Feline Immunodeficiency Virus Infection
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CHAPTER 22
Feline Leukemia Virus Infection
Jane E. Sykes and Katrin Hartmann
Overview of Feline Leukemia Virus Infection
First Described: Scotland, 1964 (Jarrett et al.)1
Cause: Feline leukemia virus (family Retroviridae, subfamily
Orthoretrovirinae, genus Gammaretrovirus)
Affected Hosts: Domestic and some wild Felidae
Geographic Distribution: Worldwide
Mode of Transmission: Prolonged close contact with salivary
secretions; to a lesser extent biting, transplacental trans-
mission, transmission through milk, and through blood
transfusion
Major Clinical Signs: Lethargy, fever, pallor, stomatitis, signs
of underlying lymphoma or leukemia, signs of immune-
mediated disorders or opportunistic infections
Differential Diagnoses: FIV infection is the primary differen-
tial diagnosis for cats with signs of immunosuppression;
feline calicivirus infection, or FIV infection are differen-
tial diagnoses for cats with stomatitis; primary immune-
mediated disease and other bone marrow diseases are
differential diagnoses for cats with immune-mediated
diseases and cytopenias
Human Health Significance: FeLV does not infect humans.
Etiology and Epidemiology
FeLV is an enveloped RNA virus that belongs to the genus Gam-
maretrovirus of the family Retroviridae. FeLV infection remains
an important cause of mortality in domestic cats through its
ability to cause immune suppression, bone marrow disorders,
and hematopoietic neoplasia. FeLV also causes disease in wild
felids such as the highly endangered Iberian lynx.2
FeLV infection progresses more rapidly than FIV infection
and is more pathogenic, so most cats that develop progressive
infections ultimately die of FeLV-related disease. However, in
contrast to FIV infection, many cats in the early state of FeLV
infection regress to a permanent state of viral latency (“regres-
sive infection”). It is possible that some cats, after exposure
to a low dose of FeLV, may eliminate the infection altogether
(“abortive infection”), although this appears to be a rare out-
come. Regardless, a positive test result for FeLV infection in an
apparently healthy cat does not always imply that FeLV-related
disease and mortality will occur.
The structure of FeLV is similar to that of FIV, except that
the capsid is icosahedral rather than cone shaped (Figure 22-1).
There are three main subtypes of FeLV: FeLV-A, FeLV-B, and
224
FeLV-C. Each subtype uses a different receptor to enter cells
(Table 22-1). All cats infected with FeLV-B and FeLV-C are co-
infected with FeLV-A, and only FeLV-A is transmitted between
animals. FeLV-B and FeLV-C are more pathogenic than FeLV-
A. FeLV-B arises through recombination of FeLV-A proviral
DNA with endogenous FeLV sequences present in host cellu-
lar DNA.3 FeLV-C arises from accumulation of mutations or
insertions in the env (SU) gene of FeLV-A.4,5 The FeLV subtype
influences the clinical expression of disease (see Table 22-1).
For example, FeLV-C is associated with nonregenerative ane-
mia. Even within an FeLV subtype, mutations in the SU and
the LTR regions of the viral genome affect disease outcome.6,7
An additional subtype, FeLV-T, has been associated with
immunodeficiency.
Transmission of FeLV-A primarily results from close con-
tact with salivary secretions, such as through licking, mutual
grooming, and shared food and water dishes. Other routes of
transmission, such as by biting, blood transfusion, in milk, and
possibly by fleas, can also occur.8-10 The virus survives poorly
outside the cat and is readily inactivated by disinfectants, soap,
and desiccation. The overall prevalence of FeLV infection
has declined over the past two decades with more extensive
testing and vaccination. Before the institution of widespread
testing and vaccination, more than 30% of cats in some catter-
ies were infected.11 In the early 1990s, the overall prevalence
of infection was 13% in nearly 28,000 sick cats from North
Matrix
Capsid protein (p27)
Integrase
Reverse transcriptase
RNA
Surface glycoprotein (SU or gp70)
FIGURE 22-1  Structure of feline leukemia virus. Gammaretroviruses contain two
identical strands of RNA and associated enzymes, which include reverse transcriptase,
integrase, and protease, packaged into a core composed of the capsid protein (p27) with a
surrounding matrix, all enclosed by a phospholipid membrane envelope derived from the
host cell. The envelope contains a gp70 glycoprotein and the transmembrane protein p15E.

TABLE 22-1
Host Cellular Receptors Involved in FeLV Infection
FeLV Subtype Receptor Receptor Function
FeLV-A FeTHTR1* Thiamine transporter protein
FeLV-B FePit1 or FePit2† Inorganic phosphate transporter
protein
FeLV-C FLVCR‡ Heme transporter protein
*Mendoza R, Anderson MM, Overbaugh J. A putative thiamine transport protein is a receptor for feline leukemia virus subgroup A. J Virol
2006;80(7):3378-3385.
†Anderson MM, Lauring AS, Robertson S, et al. Feline Pit2 functions as a receptor for subgroup B feline leukemia viruses. J Virol 2001;75(22):
10563-10572.
‡Keel SB, Doty RT, Yang Z, et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319(5864):
825-828.
America that were at risk for exposure.12 The prevalence was
just 7% in a similar population of approximately 1400 cats
in 2006.13 The prevalence of FeLV infection in European cats
has also declined markedly.14 Currently, the overall prevalence
of infection in mixed populations of cats is 1% to 6%.13,15-19
Cats with access to the outdoors; those that have contact with
other cats; cats that are male, aggressive, or intact; and cats
that are co-infected with FIV are at increased risk of FeLV
infection.12,13,15,17 Male cats are less strongly predisposed to
FeLV infection than to FIV infection, and in some studies,20
no male predisposition has been recognized. Adult cats are
more likely to be infected with FeLV than cats aged less than 6
months,13,17 but the median age of cats infected with FeLV is
3 years,15 which is lower than that for FIV. This reflects a) the
greater degree of pathogenicity of FeLV than FIV and its ability
to significantly reduce lifespan; and b) the phenomenon of age-
related resistance to FeLV, whereby exposure of cats that are
less than 4 months of age to the virus is much more likely to lead
to progressive infection than exposure of adults.21 When 12- to
16-week old kittens are exposed to a multicat household with
endemic FeLV infection, between 60% and 70% of kittens will
become infected within a 5-month time period. In contrast, less
than 5% of cats aged 6 months or older will become infected
over the same time period; over a 2-year period, 40% to 50%
of these adult cats will become infected. Infection of adult cats
can also still occur with exposure to high doses of the virus or
when there is underlying host immune compromise.
Clinical Features
Signs and Their Pathogenesis
The outcome of FeLV infection is extremely variable and
depends strongly on the virus strain involved, the challenge
dose, the route of inoculation, and factors that influence host
immune function such as age, genetics, co-infections, stress,
and treatment with immunosuppressive drugs. After oronasal
exposure to the virus, the virus replicates in oral lymphoid tis-
sue and then circulates in a few monocytes and lymphocytes
within peripheral blood. Some cats develop systemic signs, such
CHAPTER 22  Feline Leukemia Virus Infection 225
Comments
Present in all cats with FeLV; transmitted exog-
enously
Results from recombination between FeLV-A
and feline endogenous FeLV-related retrovirus
sequences; may accelerate development of lym-
phoma or enhance ­neuropathogenicity
Arises from point mutations in FeLV-A env gene;
­associated with nonregenerative anemia
as fever, lethargy, and/or lymphadenopathy, during this period.
A small number of infected lymphocytes then travel to the bone
marrow, where the virus infects rapidly dividing precursor cells
and subsequently lymphoid and epithelial cells throughout the
body. This infection of the bone marrow is considered a critical
step in the pathogenesis of FeLV infection. Once infection of
epithelial cells in the salivary glands occurs, the virus is shed in
massive quantities in saliva; low quantities of virus can also be
shed in urine and feces.
Possible outcomes of infection with FeLV are shown in
Figure 22-2. The immune system of some infected cats sup-
presses viral replication within a few weeks after infection,
before significant infection of the marrow occurs. These cats
develop a regressive infection, whereby proviral DNA is present
in the host cell genome but production and shedding of virus no
longer occurs (see Chapter 21). This may occur after the initial
period of viremia, or viremia may never be detectable.22 Regres-
sive infection can persist for life and may be reactivated with
immunosuppression, such as might occur during pregnancy or
following treatment with immunosuppressive drugs.23 Later in
life, an unknown percentage of cats with regressive infections
may develop FeLV-negative malignancies as a result of integra-
tion of viral DNA within host cellular oncogenes. Most cats
with regressive infection, however, never develop clinical signs
related to FeLV infection. Viral genome sequences may eventu-
ally be incompletely replicated, and as a consequence, reactiva-
tion of virus replication will become impossible in some cats
over time. In abortive infections, no viremia occurs after infec-
tion, and virus cannot be detected using any method. Cats with
abortive infections have been exposed to low doses of FeLV,
and although they fail to develop viremia, they will develop
antibodies to the virus.24 Some cats that never test antigen-
positive may have focal infections, that is, evidence of proviral
DNA in some tissues but not in blood or bone marrow.9 Cats
develop progressive infection once involvement of the marrow
is established and cellular destruction by the virus exceeds the
ability of the host’s immune system to suppress viral replica-
tion. Persistent viremia and progressive FeLV-related disease
result (Box 22-1).
226 SECTION 1  Viral Diseases

FeLV exposure
All antigen tests –
BOX 22-1
All PCR tests –
Clinical Outcomes of Progressive FeLV Infection
Week 1
Variable
Neoplasia, especially lymphoma, leukemia and fibrosarco-
Strong
immune response immune response mas (with FeSV)
Opportunistic infections
Abortive infection Focal/Atypical infection Pure red cell aplasia
Virus eliminated Antigen – or transiently + Aplastic anemia, myelodysplasia, or myelofibrosis
All antigen tests – Blood PCR – Immune-mediated disease (immune-mediated hemolytic
All PCR tests – Tissue proviral PCR + anemia, thrombocytopenia, glomerulonephritis, polyar-
Week 3 thritis, uveitis)
Peripheral lymphadenopathy
p27 ELISA antigen +
IFA antigen – Neurologic disease (e.g., anisocoria, urinary incontinence)
Good All PCR tests + Poor/Absent Reproductive failure
immune response immune response Gastrointestinal disease (uncommon)
Other (e.g., osteochondromatosis, cutaneous horns)
Regressive infection Progressive infection
All antigen tests – All antigen tests + FeSV, feline sarcoma virus.
Proviral PCR Weak + All PCR tests +
RT-PCR – Immunosuppression

FeLV-related disease
FIGURE 22-2  Outcomes of infection with FeLV. In the first week of infection, cats
test negative with all antigen assays (i.e., p27 ELISA and IFA assays), RT-PCR for viral RNA,
and PCR for proviral DNA. Rarely, cats exposed to low levels of virus eliminate the infec-
tion before or at this time point (abortive infection). Soluble antigen assays and proviral
PCR assays become positive from about the third week after infection, and virus is shed
in saliva, tears, urine, and feces. After this time point, productive viral infection is sup-
pressed by the host immune response (regressive infection) or the virus multiplies rapidly
in the bone marrow and a progressive infection occurs, which is characterized by persis-
tently positive antigen ELISA, IFA, viral RNA, and proviral DNA assay results. Cats with focal A
or atypical infection are never or transiently antigen positive and test negative using PCR
assays on blood or bone marrow, but some tissues (such as the mammary gland or blad-
der) are positive with proviral PCR. The latter occurs in only a small percentage (e.g., <
5%) of infected cats.

Opportunistic Infections
Opportunistic infections may develop in FeLV infections as a
result of myelo suppression or an acquired cell-mediated immu-
nodeficiency. The immunosuppressive properties of FeLV are
not fully understood but have been linked in part to the viral
envelope peptide, p15E, which inhibits T and B cell function,
inhibits cytotoxic lymphocyte responses, alters monocyte mor-
phology and distribution, and has been associated with impaired
cytokine production and responsiveness.25-28 Kittens with pro-
gressive FeLV infection have impaired T cell and, to a lesser B
extent, B cell function.29-32 Infected cats may develop lymphope-
nia, thymic atrophy, and depletion of lymphocytes within lymph FIGURE 22-3  Opportunistic infections in cats with progressive FeLV infection. A,
node paracortical zones. CD4+ T cell malfunction may contrib- Ulceration of the nasal planum in a 6-month-old male neutered domestic medium hair
ute to a decreased humoral and cell-mediated immune response cat with progressive FeLV infection as defined by positive ELISA antigen and IFA assays
on peripheral blood. Stomatitis was also present, and feline calicivirus infection was sus-
in affected cats,33,34 and the response to vaccination may also be
pected. B, Severe nasal cryptococcosis in a Siamese cat from a cattery with endemic FeLV
impaired. Impaired neutrophil function compounds the effect of infection.
neutropenia in some infected cats.35-37 Opportunistic infections
that result include bacterial infections of the upper and lower
urinary tract, hemoplasmosis, upper respiratory tract infections, but clinical signs are more severe and refractory to therapy.
feline infectious peritonitis, chronic stomatitis, toxoplasmosis, Infection with FeLV is an established risk factor for hemoplasma
dermatophytosis, and cryptococcosis (Figure 22-3). The preva- infection. In one study, it was also a risk factor for Bartonella
lence of some of these infections in cats with FeLV infection does henselae infection, but no evidence of clinical disease was identi-
not differ significantly from that in cats not infected with FeLV, fied in the cats infected with B. henselae.38

Neoplasia
FeLV causes neoplasia in cats primarily as a result of insertional
mutagenesis, by which the virus activates proto-oncogenes (espe-
cially c-myc, but also others such as flit-1) or disrupts tumor
suppressor genes.39 The most common types of neoplasia in cats
infected with FeLV are lymphoma and leukemia. Cats infected
with FeLV are more than 60-fold more likely to develop lym-
phoma than cats not infected with FeLV; this compares with
approximately 5-fold for FIV.40 Lymphoma or leukemia can
be detected in nearly one quarter of cats with progressive FeLV
infection at necropsy.41 In the 1980s, as many as 70% of cases of
feline lymphoma were associated with FeLV infection, but with
improved control measures and vaccination, the vast majority
of cats (more than 80% to 90%) seen at veterinary clinics with
lymphoma now test negative for FeLV antigen.42-44 The most
common types of lymphoma in cats infected with FeLV are
thymic (mediastinal), multicentric, spinal, renal, or ocular lym-
phoma. FeLV-associated lymphomas are mostly of T-cell origin,
but B cell lymphoma can also occur. In contrast, FIV-associated
lymphomas tend to be of B-cell origin.42 Approximately 80%
of cats with thymic lymphoma test positive for FeLV antigen,
whereas fewer than 10% of cats with gastrointestinal lymphoma
are FeLV antigen-positive. Large granular lymphoma is rarely
associated with FeLV infection.45 Cats with thymic lymphoma
typically develop clinical signs of lethargy, tachypnea, and some-
times regurgitation. Most FeLV-positive cats with lymphoma are
less than 4 years of age.14
Cats with regressive infection appear to be at greater risk for
development of lymphoma than cats that were never exposed
to FeLV. FeLV-negative cats that live with cats that test posi-
tive for FeLV antigen have a more than 40-fold increased risk
of lymphoma compared to that expected without exposure
to FeLV.46 Several studies have investigated the prevalence of
FeLV proviral DNA in tumor cells from cats that test negative
for FeLV antigen. Some studies (including more recent studies
that used real-time PCR assays) found no evidence of FeLV pro-
viral DNA in feline lymphomas.14,47,48 In older studies that used
conventional PCR, more than 20% of antigen-negative lym-
phomas tested positive.49,50 Additional studies from a variety
of geographic locations that use multiple real-time PCR assays
are required to clarify the role that FeLV plays in lymphomas
and leukemias that develop in cats that test negative for FeLV
antigen.
FeLV is responsible for the majority of myelogenous leuke-
mias, erythroleukemias, megakaryocytic, and lymphoid leuke-
mias in cats, although not all cats with these tumors test positive
for FeLV antigen. Most FeLV-associated leukemias are acute.
FeLV infection can underlie chronic eosinophilic leukemia,51
chronic myelomonocytic leukemia,52 and chronic lymphocytic
leukemia. However, most cats with chronic lymphocytic leuke-
mia are seronegative for FeLV antigen.
FeLV infection can result in the development of multiple
fibrosarcomas in young cats. These occur when FeLV-A recom-
bines with cellular oncogenes (such as c-fes, c-fms, or c-fgr) to
form feline sarcoma viruses (FeSV). These viruses then develop
mutations in these oncogenes that, when reinserted into cellular
DNA, cause malignant transformation.53,54 To replicate, FeSV
requires the presence of FeLV-A, which supplies proteins such as
those encoded by the env gene. Thus, all cats infected with FeSV
test positive for FeLV antigen. FeSV-associated fibrosarcomas
are characterized by multifocal, locally invasive, often ulcerated
cutaneous masses that metastasize readily to the lung and other
CHAPTER 22  Feline Leukemia Virus Infection 227
BOX 22-2
Mechanisms of Anemia in Cats Infected with FeLV
Decreased RBC Production
Pure red cell aplasia (FeLV-C)
Aplastic anemia
Leukemia (myelophthisis)
Myelofibrosis
Anemia of inflammatory disease
RBC Loss
Thrombocytopenia secondary to immune-mediated
or bone marrow disease
Increased RBC Destruction
FeLV-associated IMHA
Co-infection with hemoplasmas
IMHA, immune-mediated hemolytic anemia.
sites with a poor prognosis. FeLV-FeSV DNA sequences have
been detected in some uveal melanomas from cats,55 but other
studies have failed to identify an association between FeSV and
ocular melanomas or sarcomas in cats.56,57 FeLV infection does
not appear to be important in the pathogenesis of solitary fibro-
sarcomas or injection-site sarcomas in cats.58,59
Other tumor types that are variably associated with FeLV
infection are feline olfactory neuroblastomas, cutaneous horns,
and osteochondromatosis (multiple cartilaginous exostoses).60
Feline olfactory neuroblastomas are rare and aggressive tumors
of the nasal cavity that can lead to signs of rhinosinusitis and
neurologic signs. Osteochondromatosis is characterized by
benign cartilage-capped exostotic growths that arise from the
surface of a bone, which can cause pain, lameness, disfigure-
ment, and paresis due to spinal cord compression. Cutaneous
horns occur as a result of benign keratinocyte hyperplasia.
Anemia and Bone Marrow Disorders
Multiple mechanisms can lead to anemia in cats infected with
FeLV (Box 22-2). Approximately 90% of FeLV-associated ane-
mias are nonregenerative.61 A variety of bone marrow disorders
lead to decreased red blood cell production. FeLV-C infection
results in pure red cell aplasia, a severe nonregenerative ane-
mia associated with erythrocyte macrocytosis and depletion of
erythroid precursors in the bone marrow. This occurs because
FeLV-C binds to and interferes with a heme exporter protein,
which results in subsequent heme toxicosis to the developing
erythrocyte.62 The presence of macrocytosis in the absence of
reticulocytosis should thus raise suspicion for FeLV infection.
FeLV infection can also lead to aplastic anemia in some cats,
which is a deficiency in all cell lineages (platelets, myeloid, and
erythroid) in the bone marrow, and replacement of the bone
marrow space by adipose tissue. Anemia and bone marrow
dysfunction may also result from myelophthisis secondary to
leukemia, myeloid and/or erythroid dysplasia, or myelofibrosis
(progressive replacement of the marrow with collagenous con-
nective tissue) (Figure 22-4). Myelodysplasia is characterized by
disordered maturation of marrow precursors, which in some
228 SECTION 1  Viral Diseases
A phthisis, or maturation arrest at the myelocyte or metamyelo-
B may occur secondary to central nervous system (CNS) neo-
FIGURE 22-4  Bone marrow abnormalities in cats with FeLV infection. A, Bone mar-
row aspirate cytology from a 6-month old male neutered domestic shorthair cat that was
evaluated for lethargy and fever. Acute leukemia, marked erythroid aplasia, marked gran-
ulocytic dysplasia, and increased eosinophilopoiesis is present. Megakaryocytic dysplasia
was also identified. The G:E ratio was approximately 2950:1 with marked erythroid aplasia.
Wright’s stain. B, Bone marrow aspirate cytology from a 1-year-old male neutered domes-
tic shorthair. Severe megaloblastic erythrodysplasia and mild megakaryocytic dysplasia
were identified. The myeloid series is present but markedly decreased in all stages; the
myeloid to erythroid ratio was estimated at 1:6.5 (compare with A). Nuclear to cytoplasmic
asynchrony (megaloblastic change) is present, with immature nuclei in cytoplasm with
hemoglobinization. Mitotic figures are present in moderate numbers and are seen in the
late stages of the erythroid series (arrows).
cases precedes emergence of leukemia. FeLV infection under-
lies many myelodysplastic syndromes (MDS) and leukemia in
cats.63 However, over the past two decades, 50% of 28 cats
with myelodysplasia and 44% of 41 cats with leukemia seen
at the University of California, Davis, tested negative for FeLV
antigen in peripheral blood, and only 36% of 34 cats with dys-
myelopoiesis seen at the University of Minnesota were positive
for FeLV antigen.64 Several different classification systems have
been used to describe feline MDS.64 The French-American-
British (FAB) classification scheme used for humans has been
modified for dogs and cats. This divides MDS into 2 groups:
1) MDS with refractory cytopenia (less than 6% myeloblasts
in the bone marrow, MDS-RC) and 2) MDS with excessive
numbers of myeloblasts (6% to 30% myeloblasts, MDS-EB).
The presence of more than 30% blasts indicates leukemia. The
most common form of MDS in cats is MDS-EB.64 Regressive
FeLV infections have been detected by proviral DNA PCR in
some cats with bone marrow disorders,65 but the relationship
between the presence of proviral DNA and the pathogenesis of
disordered marrow function is unclear.
Anemia in cats with FeLV infection may also result from
anemia of inflammatory disease, which can be triggered by
opportunistic infections or neoplastic disease. Erythrocyte
destruction occurs in some FeLV-infected cats as a result of
secondary immune-mediated hemolytic anemia (IMHA) or co-
infection with hemoplasmas (see Chapter 41). Hemorrhage as
a result of thrombocytopenia or defective platelet function may
also contribute to anemia. Mechanisms of thrombocytopenia
include immune-mediated thrombocytopenia (ITP) and bone
marrow dysfunction. Similarly, neutropenia can result from
myeloid hypoplasia, myelofibrosis, myelodysplasia, myelo-
cyte stages. In some cases, peripheral neutropenia accompanied
by myeloid hyperplasia reflects underlying immune-mediated
neutropenia.
Immune-Mediated Disorders
In addition to immune-mediated cytopenias, other immune-
mediated disorders that can occur in association with progressive
FeLV infection are glomerulonephritis,66 uveitis,67 and polyar-
thritis.68 Circulating immune complexes have been detected in
infected cats.69-71 Because primary immune-mediated disorders
are otherwise rare in cats, retrovirus testing is essential in cats
that are diagnosed with these conditions before immunosuppres-
sive drug treatment is initiated.
Neurologic Disorders
Neurologic disorders in cats with progressive FeLV infections
plasia, opportunistic infections, or FeLV infection itself. The
envelope protein of FeLV may be neurotoxic.72,73 Anisocoria,
mydriasis, Horner’s syndrome, and urinary incontinence can
occur. FeLV infection may be one of the most frequent causes
of urinary incontinence in cats, an otherwise uncommon condi-
tion. A myelopathy has been described in FeLV-infected cats
that showed various neurologic signs such as disorientation,
lethargy, increased vocalization, progressive ataxia, paresis,
paralysis, hyperesthesia, urinary incontinence, recurrent consti-
pation, and anisocoria with diminished pupillary light reflexes.74
At necropsy, abundant FeLV antigen was detectable within neu-
rons, endothelial cells, oligodendroglia, and astrocytes within
the spinal cord.74
Gastrointestinal Disease
Uncommonly, FeLV causes enteritis that clinically and histo-
logically resembles that caused by feline panleukopenia virus
(FPV), except that lymphoid depletion is absent.75 Clinical signs
include vomiting, acute or chronic diarrhea that may be hemor-
rhagic, inappetence, weight loss, and dehydration. FeLV-infected
cats with “panleukopenia-like syndrome” have intestinal crypt
destruction and pancytopenia that results from myeloid destruc-
tion (Figure 22-5).76 Although now very rare, co-infections with
FeLV and FPV can also occur, and the 2 viruses may enhance
each other’s pathogenicity.77 Co-infections with FeLV and other
enteric viruses, such as feline coronavirus, also occur.78 In most
cases, diarrhea in cats infected by FeLV results from an etiology
other than FeLV alone.

FIGURE 22-5  Histopathology of a colonic biopsy specimen from a 6-year-old intact
male FeLV+ domestic shorthair with a 1-week history of anorexia, weight loss, lethargy,
fever, and bloody mucoid diarrhea. Severe, diffuse, plasmacytic colitis was present with
crypt epithelial necrosis and regeneration. One crypt is dilated, lined with attenuated
epithelium, and contains necrotic debris (red arrow). An adjacent crypt shows evidence
of regeneration (blue arrow). A tissue gram stain showed a mixed population of bacteria
on the luminal surface, but not within crypts. A CBC showed thrombocytopenia (22,000
platelets/µL) and neutropenia (2700 neutrophils/µL) with neutrophil toxicity. A fecal par-
vovirus antigen test was negative, and bone marrow aspirate cytology showed evidence of
myelodysplasia. Remarkably, this cat lived another 3 years with transfusion support, but
ultimately was euthanized because of acute granulocytic leukemia with secondary sepsis.
Reproductive Disorders
Transplacental spread of FeLV can lead to fetal resorption, abor-
tion, neonatal death, and fading kitten syndrome.76 Fetal loss
may also result from secondary endometritis. Kittens infected in
late gestation or after birth develop thymic atrophy, failure to
nurse, dehydration, lethargy, and death within the first 2 weeks
of life.
Physical Examination Findings
Physical examination findings in cats with progressive FeLV
infection vary dramatically depending on the stage of infec-
tion and the secondary disease process present. There may be
no abnormalities, or cats may show signs of lethargy, pyrexia,
mucosal pallor, petechial hemorrhages, dehydration, peripheral
lymphadenomegaly, thin body condition, stomatitis, subcutane-
ous abscesses, or upper respiratory tract disease. Anemic cats
may have a hemic murmur, tachypnea, or tachycardia, or they
may be icteric. Cats with severe anemia can be laterally recum-
bent or comatose and hypothermic. Cats with thymic lymphoma
may be tachypneic and have decreased lung and cardiac sounds
on thoracic auscultation as a result of malignant pleural effusion,
or heart sounds may be displaced caudally. Decreased compress-
ibility of the cranial thorax may also be detected. Splenomegaly,
hepatomegaly, renomegaly, intestinal masses, and/or abdominal
lymph node enlargement may be detected on abdominal palpa-
tion of cats with multicentric lymphoma. Neurologic signs are
detected infrequently when compared with signs of anemia or
thoracic or abdominal lymphoma and include ataxia, anisoco-
ria, and mydriasis. Although also relatively uncommon, uveitis
may be identified, or there may be other ocular abnormalities
as a result of intraocular lymphoma or co-infections with other
pathogens.
CHAPTER 22  Feline Leukemia Virus Infection 229
Diagnosis
Infection with FeLV is often diagnosed when healthy cats are
screened for infection. Screening should be performed with
ELISA or related immunochromatographic in-house assays for
free FeLV antigen in serum, because these assays are sensitive,
specific, rapid, widely available, and most well understood. The
retrovirus status of all cats should be known regardless of the
presence of absence of illness.79 The indications for retrovirus
testing are described in Chapter 21 (see Box 21-1).
Even though many cats that test positive for FeLV antigen
have no clinical signs or physical examination abnormalities, a
CBC, chemistry panel, and urinalysis should be obtained from
these cats (and at a minimum, a complete CBC with blood smear
evaluation) to assess for underlying abnormalities that could sig-
nal the presence of FeLV-related disorders. Subtle hematologic
abnormalities, such as erythroid macrocytosis or monocytope-
nia, may be present in the absence of overt clinical signs and can
signify a poorer long-term outcome. Additional diagnostic tests
indicated in infected cats that are anemic include a reticulocyte
count, Coombs’ test, and PCR assay for hemoplasmas. Bone
marrow aspiration and core biopsy are indicated in cats with
pancytopenia or persistent nonregenerative anemias.
Laboratory Abnormalities
Complete Blood Count
The CBC may be normal or show regenerative or nonregenera-
tive anemia, neutropenia, lymphopenia, monocytopenia, and/
or thrombocytopenia. Evidence of agglutination may be pres-
ent in cats with IMHA. Moderate to marked leukocytosis and
increased band neutrophils may also be present. Large numbers
of circulating blasts, megakaryocytes or dysplastic cells (such
as erythrocytes with giant Howell-Jolly bodies) can be found in
cats with leukemia or MDS. When compared with uninfected
cats, FeLV-infected cats were nearly 3.8-fold more likely to be
anemic, 5-fold more likely to be thrombocytopenic, 3.6-fold
more likely to be neutropenic, and 2.8-fold more likely to have
lymphocytosis.80
Serum Biochemical Tests and Urinalysis
Findings on serum biochemistry analysis and urinalysis are
nonspecific and reflect underlying disease processes. Hyper-
bilirubinemia and bilirubinuria may be present in cats with
immune-mediated hemolytic anemia or hemoplasmosis. Cats
with glomerulonephritis may be proteinuric. Some cats have
evidence of bacterial urinary tract infections. Urine culture and
susceptibility testing of a urine specimen obtained via cystocen-
tesis are indicated in cats with suspected urinary tract infection.
Bone Marrow Cytology and Histopathology
Both bone marrow aspirate and core biopsy specimens should
be obtained in cats with pancytopenia or nonregenerative ane-
mia. If aspirate results are not diagnostic, the core biopsy should
be submitted for interpretation. This is because bone marrow
aspirates from cats with aplastic anemia or myelofibrosis are
typically of low cellularity. Bone marrow findings in cats with
FeLV infection include evidence of neoplastic lymphoid, ery-
throid, or myeloid cells (which circulate in the peripheral blood
of cats with leukemia); myelodysplasia; hypoplasia or aplasia
of erythroid, myeloid, or megakaryocyte cell lines; erythroid,
myeloid, and megakaryocyte hyperplasia despite peripheral
cytopenias; and megakaryocyte hypoplasia. Cytochemical stains
230 SECTION 1  Viral Diseases

that identify cells of the myeloid lineage (such as alkaline phos-
phatase, peroxidase, Sudan black B, and nonspecific esterase),
immunocytochemistry, or flow cytometry using antibodies that
target cell surface cluster of differentiation (CD) molecules may
be needed to definitively identify the cell type involved in some
acute undifferentiated leukemias.
Diagnostic Imaging
Imaging findings in cats with FeLV infection reflect the underly-
ing disease process and are extremely variable. Cats with FeLV-
associated thymic lymphosarcoma have a mediastinal mass
on thoracic radiography that may be accompanied by mild to
severe pleural effusion (Figure 22-6). Abdominal sonography in
cats with multicentric lymphoma may reveal hypoechoic and
enlarged abdominal lymph nodes and enlargement, hypoecho-
genicity, or mottling of the spleen, liver, or kidneys. Increased
hepatic echogenicity can also occur with lymphoma. Intestinal
masses with loss of normal bowel wall layering may also be
detected. Splenomegaly may be detected in cats with immune-
mediated cytopenias.
Microbiologic Tests
Diagnostic assays available for FeLV infection are listed in
Table 22-2.

FIGURE 22-6  Lateral thoracic radiograph from a 5-year-old male neutered domes-
tic shorthair cat with mediastinal lymphoma 11 months after it tested positive for cir-
culating FeLV antigen. The cat was tachypneic and had muffled heart sounds that were
displaced caudally. There is evidence of pleural effusion and dorsal displacement of the
trachea. Cytologic examination of pleural fluid revealed large numbers of malignant
lymphocytes.
Antigen Assays
The initial assay of choice for diagnosis of FeLV infection is an
ELISA or a similar immunochromatographic test that detects
soluble p27 capsid protein antigen in blood. The term soluble is
used to distinguish these assays from assays such as IFA, which
detect fixed antigen within cells. In most cats, the presence of
circulating antigen correlates with viremia, although a few cats
have viremia in the absence of detectable antigen or antigenemia
in the absence of detectable viremia.79 In-practice lateral flow
assays are available that detect antigen in anticoagulated whole
blood, plasma, or serum. In the past, the use of whole blood
generated less reliable results than when plasma or serum was
used, but with new-generation tests, whole blood is considered
an acceptable alternative.79 When a choice is available, serum is
the preferred specimen. The use of tears or saliva is not recom-
mended, because errors are more likely to occur. When virus
isolation in culture was used as the gold standard, the sensitivity
of seven different assays ranged from 92.1% to 96.8%, and the
specificity ranged from 95.4% to 99.2%.81

TABLE 22-2
Diagnostic Assays Available for Feline Leukemia Virus Infection
Assay Specimen Type Target Performance
ELISA or ­similar Serum, plasma, whole FeLV p27 antigen Confirmation of positive results is recommended in healthy
immuno-­ blood cats with a second test from a different manufacturer.
chromatographic Positive antigen test results do not signify progressive
tests for soluble infection, and the assay must be repeated in 1 to 3 months
FeLV antigen or an IFA performed. False negatives can occur in the first
month of infection.
IFA Serum, bone marrow FeLV antigen in Less sensitive than ELISA. Positive results indicate infection
blood cells of the bone marrow and therefore progressive infection.
False positives may occur if nonspecific fluorescence is
interpreted as a positive result.
PCR Blood, bone marrow, FeLV RNA (RT-PCR) Sensitivity and specificity may vary between laboratories.
saliva (RT-PCR); or ­proviral DNA Never use in the absence of antigen testing. Assays that
bone marrow, tissue, have demonstrated sensitivity and specificity may be ­useful
lymph node aspirates to detect cats with regressive infection for ­elimination
(PCR) from blood donor programs, or to resolve the results
of discordant ELISA and IFA assays. False-negative test
results may occur when variant strains are present.
Virus isolation Blood, bone marrow Replication-competent Difficult, not widely available. Requires a specialized
FeLV virus ­laboratory. Used primarily as a research tool.

IFA, immunofluorescent antibody; RT, reverse transcriptase.


When ELISAs are used as screening tests, confirmation of
positive test results is recommended because of the low preva-
lence of infection in healthy cats and the higher possibility that
false-positive test results may occur. Positive test results in the
absence of FeLV antigen have the potential to occur rarely as a
result of operator error or nonspecific reactivity.82 As for FIV
infection, it is especially important to immediately confirm posi-
tive test results if they are likely to result in euthanasia or rehom-
ing for disease control purposes. There are several options to
confirm a positive test result:
• Perform another ELISA antigen test using an assay from a
different manufacturer. However, it should be remembered
that in contrast to FIV infection, cats that test truly posi-
tive for FeLV antigen early in the course of infection (i.e.,
before involvement of the bone marrow) may ultimately con-
trol the infection. Thus a single positive test result does not
imply progressive infection, even if it is immediately repeat-
able using a test from a different manufacturer. If the cat has
signs consistent with FeLV-related disease, a single positive
test result is more likely to mean that progressive infection is
present.
• Perform an IFA assay on peripheral blood smears, because
cats with positive IFA results have infection of the bone mar-
row and, with rare exceptions, are almost always progres-
sively infected. Cats that test negative with IFA assays may
be in a transient viremic phase that may result in either pro-
gressive or regressive infection, or they may have progressive
infection but the sensitivity of IFA is too low to detect it. In
this case, both ELISA and IFA assays, or the ELISA assay
alone, could be repeated in 1 to 4 months.
• Retest with ELISA 6 months later. If the antigen test remains
positive, progressive infection is likely. In some cats, antigen-
emia persists for 16 weeks before regression occurs, so the
test could be repeated earlier than 6 months (e.g., 12 weeks
later) or monthly if client finances permit so long as the cat
remains healthy.
• Perform a full CBC. If hematologic abnormalities are pres-
ent, progressive infection is likely.
Negative ELISA results can occur in the first month after expo-
sure to FeLV, before sufficient antigen is detectable in the
peripheral blood. Cats that test negative within 30 days of pos-
sible exposure to the virus should be retested 1 to 2 months
later. Because development of antibodies to FIV can take up to 2
months, it is usually most practical to retest for both viral infec-
tions 2 months after possible exposure. Kittens can be tested
at any time, because maternal antibody does not interfere with
FeLV testing.
Immunofluorescent Antibody or Immunoperoxidase Staining
IFA assays are widely offered by veterinary diagnostic labora-
tories and can be performed on fresh peripheral blood or bone
marrow. At least two fresh smears (without anticoagulant)
should be air-dried and mailed to the laboratory. IFA is less
sensitive than ELISA and, depending on the laboratory, is more
prone to false-negative and false-positive results and so is not
recommended for screening purposes. The presence of detect-
able virus using IFA in circulating blood cells indicates pro-
gressive infection more than 90% of the time. Cats with early
viremia (before the bone marrow is infected) test IFA-negative
but ELISA-positive. Cats with regressive infection test negative
with both IFA and ELISA assays (see Figure 22-2). Negative IFA
test results can occur in cats with progressive infection when
CHAPTER 22  Feline Leukemia Virus Infection 231
there are inadequate blood cells in the periphery, such as in
neutropenic cats. Performing IFA on bone marrow rather than
peripheral blood may help to overcome this problem. False-pos-
itive results can occur when inexperienced laboratory personnel
interpret nonspecific fluorescence as a positive test result. They
also have the potential to occur when the antibody conjugates
bind nonspecifically to eosinophil granules, so eosinophils must
be excluded in interpretation of fluorescing cells.83
Some cats with clinical abnormalities that strongly suggest
FeLV-related disease (such as leukemia or myelodysplasia) test
negative for circulating antigen using ELISA but have bone
marrow cells that test positive for FeLV antigen using IFA. For
example, at the author’s teaching hospital, 8 of 18 cats with
leukemia or MDS that tested negative for soluble FeLV anti-
gen had bone marrow smears that were positive using IFA; the
remainder tested IFA negative. This phenomenon may reflect
either false-positive IFA assay results, or true infection with
undetectable levels of antigen in the peripheral blood. The use
of PCR on bone marrow may help to resolve the FeLV status in
at least some of these cats.
Immunohistochemistry can also be used to detect viral anti-
gen in tissue specimens or bone marrow core biopsies, although
it may be less sensitive than IFA.
Molecular Diagnosis Using the Polymerase Chain Reaction
Several different PCR assays have been developed for detection
of FeLV nucleic acid. Currently the major clinical indications
for PCR are (1) to screen potential blood donors in conjunction
with antigen testing or (2) to test for regressive infection when
FeLV is strongly suspected as the cause of neoplasia but antigen
tests are negative.
PCR assays may detect FeLV RNA (reverse transcriptase–
polymerase chain reaction [RT-PCR]) or proviral DNA and
must be carefully designed so that they do not detect endog-
enous FeLV sequences. At the current time, PCR assays should
never be used in the absence of antigen testing in order to screen
for or diagnose FeLV infection. The clinician needs to under-
stand if the assay used detects proviral DNA, viral RNA, or
both (some laboratories run both assays), because the clinical
significance of a positive viral RNA assay (i.e., productive viral
infection) differs from that of a positive proviral DNA assay
(which suggests nonproductive viral infection for cats with neg-
ative antigen tests). After infection, RT-PCR assays may be posi-
tive several weeks before antigen tests or virus isolation become
positive,84 and depending on the assay, PCR for viral RNA can
be more sensitive than soluble antigen tests. High viral RNA
loads in blood and saliva appear to be associated with progres-
sive infection, whereas low loads may be associated with regres-
sive infection (Figure 22-7).84
PCR assays for proviral DNA can be used on blood, buffy
coats, bone marrow, or tissues of cats that test negative for FeLV
antigen. Cats with a positive proviral PCR test result but nega-
tive soluble antigen test, which in one study represented about
10% of cats with negative antigen test results, have regressive
infection.85,86 These cats are probably not infectious to other cats
but may reactivate virus shedding with severe stress or immu-
nosuppression, or transmit the virus through blood transfusion
or vertical transmission. Proviral DNA appears to be present
at much lower levels in cats with regressive infection than in
those with progressive infection.86,87 The cell types that contain
FeLV proviral DNA also may differ for antigen-negative and
antigen-positive cats. The proviral DNA of one FeLV strain was
232 SECTION 1  Viral Diseases
A B
E F
FIGURE 22-7  Viral RNA (A-D) and p27 antigen (E-H) loads in plasma (A and E), saliva (B and F), feces (C and G), and urine (D and H) in 10 cats experimentally infected with FeLV.
Each line represents observations for a single cat. Continuous lines represent data from cats with progressive infection, and broken lines represent data from cats with regressive infection.
(Modified from Cattori V, Randon R, Riond B, et al. The kinetics of feline leukemia virus shedding in experimentally infected cats are associated with outcome. Vet Microbiol 2009;133(3):292-
296, Figure 1.)
found only in the lymphocytes of antigen-negative cats, whereas
antigen-positive cats had high loads within all leukocyte types,
including lymphocytes, monocytes, and granulocytes.86,87 The
use of bone marrow or tissue specimens, rather than whole
blood, may increase sensitivity for detection of proviral DNA.
The sensitivity and specificity of commercially available
PCR assays are likely to vary with assay design, and the useful-
ness of assays offered commercially has not been published, so
until more information is available, great caution is warranted
when interpreting the results of PCR assays for FeLV infection.
Retroviruses have high rates of mutation, and the presence of
sequence variations may lead to false-negative results. False-
positive results occur in some PCR laboratories as a result of
contamination or manual loading errors, or poor assay design.
Because FeLV vaccine virus is inactivated or recombinant, it
does not replicate or integrate into the host genome, so vaccina-
tion should not lead to false-positive PCR results.
Virus Isolation
FeLV can be readily isolated in cell culture. The cell culture
supernatant can then be tested for production of FeLV using
antigen assays or viral RNA PCR. Because growth requires sev-
eral days and specialized techniques, cell culture is not routinely
used for clinical diagnosis.
Antibody Assays
Although not useful for diagnosis of FeLV infection, detec-
tion of antibody responses to FeLV infection is performed on a
clinical research basis with virus neutralization assays and IFA
assays that detect antibodies against feline oncornavirus cell
membrane–associated antigen (FOCMA).24,48,84,88,89 Positive
antibody test results in cats that test negative for FeLV antigen
indicate previous exposure followed by abortive or regressive
infection. Cats with progressive infections and high viral loads
C D
G H
usually fail to develop antibody responses.24,48,88 Antibody
responses may occur after vaccination with FeLV vaccines.
Pathologic Findings
Gross and histopathologic findings in cats with FeLV infection
usually reflect secondary disease processes. Lymphoma and evi-
dence of myelodysplasia or leukemias are the most common
findings, but opportunistic infections may also be detected.
The intestinal tracts of cats with FeLV-associated enteritis
reveal crypt cell necrosis and regeneration, lymphoplasmacytic
infiltrates, and blunting and fusion of villi (See Figure 22-5).90
Reactive lymphoid hyperplasia is also a common finding. CNS
lesions include loss of axons and dilated myelin sheaths within
the spinal cord.74 Immunohistochemical stains may be applied
to tissue sections to confirm the presence of FeLV antigen in
association with lesions.
Treatment and Prognosis
Cats with opportunistic infections and lymphoma can be suc-
cessfully treated using the same medications and supportive
treatments used for FeLV negative cats with these problems.
Opportunistic infections may require longer periods of treat-
ment or, in some cases, lifelong treatment with antimicrobial
drugs. Hemoplasma infections should be treated with doxycy-
cline (see Chapter 41). Cats with nonregenerative anemias may
require periodic blood transfusions. Serum erythropoietin con-
centrations are already high in these cats, and in many cases,
treatment with darbepoetin or human recombinant erythro-
poietin is unsuccessful but could be attempted. The efficacy of
treatments such as filgrastim is not known and may be com-
plicated by the formation of antibodies (see Chapter 7). The
management of cats with stomatitis is discussed in Chapter 21.
In general, glucocorticoids and other immunosuppressive drugs

TABLE 22-3
Suggested Medications for Treatment of Cats with Feline Leukemia Virus Infection
Drug Dose Route
Zidovudine (AZT) 5 mg/kg PO
Feline recombinant interferon omega 1 million U/kg SC
Human recombinant interferon alpha 1 to 50 U/cat PO
should be avoided unless immune-mediated cytopenias are sus-
pected. Some cats with FeLV-associated IMHA respond well to
treatment with glucocorticoids, and glucocorticoid treatment
may be unavoidable.
Antiviral agents and immunomodulators are of limited ben-
efit for treatment of cats with FeLV infections (see Chapter 7
for more information) (Table 22-3). The use of feline recom-
binant IFN-ω improved clinical scores and survival times in
cats with FeLV infection over a short time period (2 months) in
one study.91 Beneficial outcomes have also been described after
treatment with low-dose oral human recombinant IFN-α. Zid-
ovudine (AZT) has not performed as well for treatment of sick
cats with FeLV infections as it has for those with FIV infections,
and the results of some studies have shown minimal benefit. In
other studies, AZT improved oral cavity inflammation, reduced
antigenemia, and prolonged life span in naturally and experi-
mentally infected cats with FeLV infection.92,93 Controlled stud-
ies of the efficacy of other treatments, such as lymphocyte T-cell
immunomodulator (T-cyte Therapeutics, Inc.) and acemannan,
are required. Antivirals that show promise for treatment of
FeLV infection include fozivudine (which is closely related to
AZT) and the integrase inhibitor raltegravir, which has been
used to treat gammaretrovirus infections in humans and inhibits
FeLV replication in cell culture.94 The safety of these drugs in
cats remains to be determined.
Cats infected with FeLV should be housed indoors to prevent
spread of infection to other cats. Indoor housing also minimizes
exposure of infected cats to other opportunistic pathogens.
Raw food diets should not be fed. Survival may be prolonged
in low-stress environments, so provision of space, adequate lit-
ter boxes, management of co-infections, and a proper diet are
important. Vaccines administered for prevention of respiratory
viruses and FPV should be inactivated. Some FeLV-infected cats
may not respond as well to vaccination as noninfected cats.
Prognosis
Survival times vary considerably depending on the stage of
infection, host immunity, and the strain of FeLV involved. Nev-
ertheless, virtually all cats that are progressively infected with
FeLV go on to develop FeLV-related disease within 5 years of
diagnosis.95 A comparison of more than 800 FeLV-infected cats
and 8000 controls revealed a median survival time of 2.4 years
for FeLV-infected cats versus 6.3 years for controls.79 Many
progressively infected cats, especially adult cats, may live for
several years with a good quality of life, and so euthanasia is
not recommended on the basis of a positive FeLV test alone.
CHAPTER 22  Feline Leukemia Virus Infection 233
Interval (hours) Comments
12 Monitor CBC weekly
during treatment for
the first month, then
monthly.
q24h for 5 consecutive days
­starting on days 0, 14, and 60
24
Some cats with FeLV-associated lymphoma may have long-term
remissions when treated with standard chemotherapy protocols.
Some, but not all, studies suggest that FeLV infection is a nega-
tive prognostic indicator in cats with lymphoma.96 One of the
authors has treated a cat that initially tested positive for FeLV
antigen using ELISA and had evidence of IFA-positive, severe
megaloblastic erythroid dysplasia in the marrow and IMHA, yet
subsequently became ELISA-negative and remained in clinical
remission while being treated with glucocorticoids more than
4 years later.97 The prognosis is least favorable for cats with
leukemia, which generally survive less than a few weeks.
Immunity and Vaccination
Several parenteral vaccines are available for prevention of FeLV
infection, which include adjuvanted inactivated whole virus
vaccines; nonadjuvanted canarypox vectored virus vaccines
that incorporate the env and gag genes of FeLV (Purevax and
Eurifel, Merial); and a recombinant subunit vaccine that con-
tains p45, the nonglycosylated form of gp70 (Leucogen, Vir-
bac). The recombinant canarypox vaccine available in Europe
is a different product than that licensed for use in the United
States. Studies that have used highly sensitive PCR assays have
shown that vaccination does not produce sterilizing immunity
(that is, complete absence of viral RNA, DNA, antigenemia, and
viremia after challenge).98 In other words, cats develop regres-
sive or even progressive infections, but not abortive infections
after challenge.98 Nevertheless, vaccination with recombinant
subunit, canarypox vectored, and whole virus vaccine prevented
progressive infection in 87%, 78%, and 44% of cats when com-
pared with controls, respectively. One vaccine protected 83% of
cats against antigenemia after challenge as long as 2 years after
vaccination.99
In summary, no vaccine provides 100% protection against
FeLV infection, and even when protection against progressive
infection occurs, regressive infections still occur. However,
because vaccination protects cats from progressive infection, it
is indicated for all at-risk cats, such as those with outdoor expo-
sure or those that reside in households with other FeLV antigen-
positive cats. The American Association of Feline Practitioners
(AAFP) highly recommends that all FeLV antigen-negative kit-
tens be vaccinated for FeLV, because of the potential that some
of these kittens may escape or become outdoor cats, even when
the intention of the owner is to keep the kitten indoors.100 Vac-
cination is also recommended for cats entering shelters that are
likely to be housed with other cats.79 Two doses are given in
234 SECTION 1  Viral Diseases
the left pelvic limb as distally as possible, 3 to 4 weeks apart
from 8 to 9 weeks of age, followed by a booster at 1 year and
then every 1 to 3 years thereafter, although more information
is required on the duration of immunity for FeLV vaccines. The
European Advisory Bureau on Cat Diseases (ABCD) suggests a
booster every 2 to 3 years for cats older than 3 to 4 years of age,
in light of the lower susceptibility of adult cats to infection.101
Annual boosters are required for recombinant vaccines. Acutely
ill cats should not be vaccinated, but it is acceptable to vacci-
nate cats with chronic diseases, such as chronic kidney disease.
Testing for FeLV should be performed before each booster if
exposure to FeLV was likely before the booster was required
(which should apply for most cats vaccinated for FeLV). Vac-
cination with FeLV vaccines has been associated with injection-
site sarcomas, so only cats that are likely to be exposed should
be vaccinated.
Prevention
Prevention of FeLV infection involves indoor housing of cats
away from other cats infected with FeLV, testing and removal
(or separation) of cats that test antigen-positive from other
CASE EXAMPLE
Signalment: “Hamster” a 6-year-old female spayed domestic
shorthair from Fairfield, CA.
History: Hamster was brought to the University of California,
Davis, Veterinary Medical Teaching Hospital for the
problems of fever, lethargy, anorexia, diarrhea, and
vomiting. The diarrhea had been present for 3 days and
was greenish, mucoid, and occurred once to twice daily.
The owner reported that Hamster had vomited yellow fluid
at least three times during this period and was completely
inappetent. Her urination habits had not changed. Hamster
was taken to a local veterinary clinic on the first day of illness,
where a physical examination revealed pyrexia (105.1°F or
40.6°C). She was treated with clavulanic acid–amoxicillin
and a single intramuscular injection of enrofloxacin but
there was no clinical improvement. Hamster had tested
positive for FeLV antigen 1.5 years ago, after which time she
had been housed exclusively indoors. She lived with some
caged birds and three other FeLV-positive cats. None of
the other cats were ill. Hamster played with elastic bands,
but there was no known toxin exposure and no recent
changes in her environment. Her diet usually consisted of a
commercial dry cat food.
Physical Examination:
Body Weight: 6.9 kg
General: Quiet, alert, responsive, hydrated, T = 104.4°F
(40.2°C), HR = 220 beats/min, RR = 36 breaths/min, mucous
membranes pink, CRT = 1 s. Haircoat unkempt.
Eyes, Ears, Nose, and Throat (with Dilated Fundoscopic
Examination): The only abnormality noted was mild
gingivitis.
Musculoskeletal: Body condition score 7/9. Normal
ambulation was present.
cats, vaccination, and proper screening of blood donor cats
with soluble antigen assays and PCR. Routine hand-washing
precautions are indicated for hospitalized cats, and precau-
tions should be taken (such as the wearing of gloves) to protect
FeLV-infected cats from nosocomial pathogens. Fomites such
as food and water bowls and litter boxes should not be shared
between FeLV antigen-negative cats and cats with unknown or
antigen-positive FeLV status. Neutering can prevent roaming
and reduce the likelihood of FeLV infection. When a cat from
a multicat household tests antigen-positive, all cats should be
tested and retested and positive cats should be separated from
other cats if possible. Euthanasia is recommended for sick,
FeLV-positive cats that enter shelters.101 People who adopt cats
that have unknown retrovirus status from shelters should be
educated about the disease and the need for quarantine and test-
ing after adoption.
Public Health Aspects
Although FeLV can replicate in human cell culture lines, no con-
clusive evidence of natural infection with FeLV has ever been
detected in humans.
Cardiovascular and Respiratory: No clinically significant
abnormalities were detected.
Gastrointestinal and Urogenital: The cat resented palpation
of her cranial abdomen. The perianal region was stained
with green fecal material.
Lymph Nodes: All lymph nodes were <1 cm in diameter.
Laboratory Findings:
CBC:
HCT 24.6% (30%-50%).
MCV 48.4 fL (42-53 fL), MCHC 32.1 g/dL (30-33.5 g/dL)
Reticulocytes 7800 cells/µL
WBC 11,470 cells/µL (4500-14,000 cells/µL)
Neutrophils 10,438 cells/µL (2000-9000 cells/µL)
Band neutrophils 459 cells/µL
Lymphocytes 344 cells/µL (1000-7000 cells/µL)
Monocytes 229 cells/µL (50-600 cells/µL)
Platelets 122,000 platelets/µL (180,000-500,000 platelets/
µL). The neutrophils showed slight toxicity, and there
were a few macroplatelets
Serum Chemistry Profile:
Sodium 148 mmol/L (151-158 mmol/L)
Potassium 3.8 mmol/L (3.6-4.9 mmol/L)
Chloride 110 mmol/L (117-126 mmol/L)
Bicarbonate 18 mmol/L (15-21 mmol/L)
Phosphorus 2.9 mg/dL (3.2-6.3 mg/dL)
Calcium 9.5 mg/dL (9.0-10.9 mg/dL)
BUN 15 mg/dL (18-33 mg/dL)
Creatinine 1.2 mg/dL (1.1-2.2 mg/dL)
Glucose 169 mg/dL (63-118 mg/dL)
Total protein 7.0 g/dL (6.6-8.4 g/dL)
Albumin 2.6 g/dL (2.2-4.6 g/dL)
Globulin 4.4 g/dL (2.8-5.4 g/dL)
ALT 72 U/L (27-101 U/L), AST 121 U/L (17-58 U/L)
ALP 12 U/L (14-71 U/L), γ- GGT 0 U/L (0-4 U/L)
Cholesterol 104 mg/dL (89-258 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL).

Urinalysis (Cystocentesis): SGr 1.020; pH 7.0, 1+ protein, 2+
hemoprotein, 0-1 WBC/HPF, 0-2 RBC/HPF, rare rods, few
amorphous crystals.
Imaging Findings:
Plain Abdominal Radiographs: The small bowel was diffusely
gas- and fluid-filled with normal intestinal diameter. The
colon was relatively empty with a small amount of fluid. No
masses were identified. There was renal asymmetry with
poor visualization of the right kidney. Abdominal serosal
detail appeared normal.
Abdominal Sonography: The right kidney could not be
identified. The left kidney appeared normal and measured
4.2 cm in length. A small amount of fluid was present in the
colon. The remainder of the abdomen was unremarkable.
Microbiologic and Virologic Testing: Aerobic bacterial
urine culture: negative
Point-of-care ELISA serology for FeLV antigen and FIV antibody:
positive for FeLV antigen
PCR for Mycoplasma haemofelis and Candidatus Mycoplasma
haemominutum (whole blood): negative
Diagnosis: Progressive FeLV infection with suspected acute
pyelonephritis (characterized by fever, gastrointestinal
signs, bacteriuria, neutrophilia with a left shift, and mild
thrombocytopenia). Right renal agenesis (likely congenital).
Treatment: Hamster was hospitalized and treated with
intravenous fluids, enrofloxacin (5 mg/kg, slow IV, q24h) and
ampicillin (20 mg/kg, IV, q8h) (note that the parenteral use of
enrofloxacin in this cat was off-label and has the potential to
cause irreversible blindness). Her temperature normalized
within 24 hours, and there was no more vomiting or diarrhea.
After 48 hours, the hematocrit was 27.6%, neutrophil
count was 5759 cells/µL with no bands or toxicity, and
lymphocyte count was 917 cells/µL. She was discharged
from the hospital with instructions to continue antibiotic
treatment for 1 month. Two weeks after the antibiotics
had been discontinued, she was apparently healthy and a
SUGGESTED READINGS
Hartmann K. Clinical aspects of feline immunodeficiency and feline
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Levy J, Crawford C, Hartmann K, et al. 2008 American Association
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Feline Med Surg. 2008;10:300-316.
Lutz H, Addie D, Belak S, et al. Feline leukaemia. ABCD guidelines on
prevention and management. J Feline Med Surg. 2009;11:565-574.
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Her hematocrit was 38.9%, white cell count was 4620 cells/
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56. Cullen CL, Haines DM, Jackson ML, et al. The use of immuno-
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58. Ellis JA, Jackson ML, Bartsch RC, et  al. Use of immunohisto-
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237
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78. Kipar A, Kremendahl J, Addie DD, et al. Fatal enteritis associated
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81. Hartmann K, Griessmayr P, Schulz B, et al. Quality of different
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238 SECTION 1  Viral Diseases
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565-574.
CHAPTER 23
Feline Respiratory Viral Infections
Jane E. Sykes
Overview of Feline Viral Respiratory Disease
First Described in Cats: Feline herpesvirus-1 (FHV-1), 1958
(United States)1; feline calicivirus (FCV), 1957 (New Zea-
land)2; H5N1 influenza virus, 2006 (Thailand)3; pandemic
H1N1 influenza virus, 2010 (United States).4
Causes: Feline herpesvirus-1, feline calicivirus, avian and
human origin influenza A viruses
Geographic Distribution: Worldwide
Mode of Transmission: Close contact, fomite spread, to a
lesser extent aerosols
Major Clinical Signs: Serous to mucopurulent nasal and ocu-
lar discharge, sneezing, stertor, conjunctivitis, lingual or
facial ulceration, fever, inappetence, tachypnea, cough,
rarely death. Keratitis occurs with FHV-1 infection.
Differential Diagnoses: Infections with bacteria such as Borde-
tella bronchiseptica, Streptococcus spp., or Chlamydia felis;
other causes of rhinitis such as cryptococcosis, aspergillo-
sis, neoplasia, foreign bodies, chronic idiopathic feline rhi-
nosinusitis, nasopharyngeal stenosis, or nasopharyngeal
polyps; feline chronic airway disease.
Human Health Significance: Influenza virus infections have
been transmitted from humans to cats, and the potential
for zoonotic transmission exists. FHV-1 and FCV do not
infect humans.
Etiology and Epidemiology
Infectious feline upper respiratory tract disease (URTD) is a
widespread and important cause of morbidity and mortality
where large numbers of cats are housed together, especially
in overcrowded or stressful conditions.5 Multiple pathogens
are involved and co-infections are common (Table 23-1). The
most prevalent viral causes of URTD are feline herpesvirus-1
­(FHV-1) and feline calicivirus (FCV). Influenza viruses also
cause respiratory disease in cats but are relatively rarely iden-
tified. Bacterial causes of respiratory disease in cats include
Bordetella ­bronchiseptica, Chlamydia felis, and Mycoplasma
species. Streptococcus canis and Streptococcus equi subspecies
zooepidemicus can also play a role in shelter situations and cat-
teries. More information on the bacterial causes of feline URTD
can be found in Chapters 33, 34, 38, and 40.
Feline Herpesvirus-1
FHV-1 is a large, enveloped DNA virus that has a worldwide
distribution. Most cats are exposed to FHV-1 during their
lifetime. FHV-1 is an alphaherpesvirus that is closely related
to canine herpesvirus-1, and to a lesser extent, herpes simplex
virus. There is only a single serotype of FHV-1. Isolates are also
genetically similar, yet some variation in strain virulence exists.6
Using culture, FHV-1 has been detected in 0% to 39% of
cats with URTD, although in some catteries and shelters with
endemic FHV-1 infection, the prevalence may be much higher.
When sensitive PCR assays are used to detect FHV-1, prevalences
of infection that approach 100% have been detected in some
groups of cats with acute URTD.7 The prevalence of shedding by
apparently healthy cats has ranged from 0% to 10% and most
often has been lower than 2%.5,8-14 Virtually all infected cats
develop latent infection, which primarily occurs in the trigeminal
ganglia. FHV-1 DNA can also be detected in other tissues of the
head, such as the cornea and nasal cavity, but whether this is
a true state of latency or just chronic persistent infection is not
clear. Reactivation of shedding, with or without concurrent clin-
ical signs of URTD, occurs in less than half of latently infected
cats 4 to 12 days after stress.15 Examples of stressors include
transportation (such as to a veterinary clinic, boarding or breed-
ing facility, shelter, or cat show), lactation, exposure to new cats,
concurrent illness, or treatment with immunosuppressive drugs
such as glucocorticoids. The duration of shedding after reactiva-
tion ranges from 1 to 13 days (mean, 7 days).16,17 Shedding that
coincides with lactation results in infection of susceptible kittens.
FHV-1 survives a maximum of 18 hours at room tempera-
ture and is readily inactivated by drying and most disinfectants.
Because of this, transmission occurs primarily through close
contact, although fomites remain a very important mode of
transmission in crowded environments. Aerosol transmission is
of lesser importance.15,18 Aerosols generated by sneezing cats
typically travel no further than a distance of 4 to 5 feet.
Feline Calicivirus
Feline calicivirus (FCV) is a non-enveloped, single-stranded
RNA virus with a spherical capsid that is studded with cup-
shaped depressions (calici = “cup”) (Figure 23-1). Like FHV-1,
FCV commonly causes feline URTD, accounting for 10% to
over 50% of cases. The highest prevalences occur in large multi-
cat environments. Natural infection of dogs by FCV-like strains
has been reported.19 Like other RNA viruses, the genome of
FCV continually undergoes rapid mutation, which increases
the diversity of strains over time. Although considerable anti-
genic diversity exists among FCV isolates, the degree of anti-
genic cross-reactivity between them is sufficient for them to be
classified as a single serotype. Nucleotide sequence analysis also
suggests that isolates worldwide are a single, but highly diverse,
group. No correlations have been found between the genetic
composition of FCV strains, different clinical manifestations of
disease, or geographical location.
239
240 SECTION 1  Viral Diseases
TABLE 23-1
Viral Causes of Transmissible Respiratory Disease in Cats
Organism Incubation Period (days)*
Feline herpesvirus-1 2 to 6
Feline calicivirus 2 to 6
Influenza viruses Few days
*Incubation and shedding periods are approximate and may differ when co-infections or immune suppression operate.
FIGURE 23-1  Schematic of the structure of the capsid of feline calicivirus.
Infected cats can develop a persistent oropharyngeal infec-
tion (>1 month in duration) in the absence of obvious clinical
signs. This is termed the carrier state for FCV, and may result
from immune evasion through antigenic variation of the capsid
protein.20 FCV is shed continuously from the oropharynx, and
the magnitude of shedding varies with time and between indi-
vidual cats.21 Carrier cats serve as a source of infection for other
cats. In many cats, shedding terminates weeks to months after
infection, but in a few cats, shedding is lifelong. A single cat
can be simultaneously infected with multiple variants of FCV,
each derived from the original infecting strain as a result of
genetic mutation, drift, and selection pressures.20 Because of the
chronic carrier state, the prevalence of FCV infection in healthy
cats is high and ranged from 8% of household cats to 24% of
show cats.12
Environmental persistence of FCV is considerably more pro-
longed than that of FHV-1, and FCV resists routine disinfection
with quaternary ammonium compounds. Susceptibility to disin-
fectants may vary between FCV strains.22 Survival in the envi-
ronment has been demonstrated for as long as 28 days,23 and
related caliciviruses persist in a dried state for several months.24
As a result, fomites are a very important means of transmission.
FCV is also transmitted through direct contact with respiratory
secretions and through aerosols. Fleas may spread FCV through
their feces or when cats ingest fleas while grooming.25
Highly virulent FCV strains have been isolated from out-
breaks of severe systemic febrile illness in cats in the United
States and Europe known as virulent systemic disease (VSD),26-31
which was first described in California in 1998. Shelter cats
that were hospitalized in veterinary clinics have been a source
of infection in many outbreaks, and for each outbreak, the FCV
strain involved has differed. VSD has also been described in
one cat from a multiple-cat household, that is, in the absence
of an outbreak.32 Otherwise healthy, adult, vaccinated cats are
Shedding Period* Environmental Survival
1 to 2 weeks, with intermittent Less than a day
­reactivation of shedding with stress
Most <30 days, may be lifelong Up to 1 month
<2 weeks Hours
often affected, whereas kittens tend to show less severe signs.
Although infections typically spread rapidly in outbreaks,
including through fomites to pet cats of hospital staff, spread
of disease has been limited to affected clinics or shelters, with
no further spread within the community, and outbreaks resolve
within approximately 2 months once appropriate control mea-
sures are instituted.
Influenza Viruses
Pandemic H1N1 influenza viruses originated from pigs and
have caused widespread illness in humans. Natural infection
with these strains has been reported in cats from the United
States and Europe. Affected cats had signs of fever and respi-
ratory and gastrointestinal tract disease and, in some cases,
death.4,33-35 Transmission from sick humans who were in con-
tact with the cats was suspected. Cat-to-cat transmission may
also have occurred.34
Highly pathogenic avian origin H5N1 influenza virus infec-
tion, which emerged as a cause of human illness in Hong
Kong in 1997 and subsequently spread worldwide,36 has been
detected in sick and apparently healthy domestic cats and wild
felids from southeast Asia3,37,38 and central Europe.39,40 Nev-
ertheless, no evidence of infection was found in more than 170
cats where infected birds had been identified in Germany and
Austria.41 Infection of cats follows direct or indirect contact
with infected birds, especially consumption of raw poultry;
­cat-to-cat ­transmission may also occur.37
Canine H3N2 virus infection was associated with bron-
chopneumonia in shelter-housed cats in China and Korea.42
Experimentally, cats can also be infected with other influenza
viruses, including human H2N2 and H3N2, and avian origin
H7N7 and H7N3 viruses.43,44 Widespread evidence of exposure
to pandemic H1N1, seasonal H1N1, and seasonal H3N2 was
found in 400 cats from Ohio in the United States.45 Seropositive
cats were 7.4 times more likely to have had respiratory illness
than seronegative cats, and twice as likely to have had nonrespi-
ratory illness than seronegative cats. More information on influ-
enza viruses can be found in Chapter 17.
Clinical Features
Signs and Their Pathogenesis
After direct or indirect contact with virus in respiratory secre-
tions from the conjunctiva, nasal cavity and oropharynx, FCV
and FHV-1 replicate within lymphoid and epithelial cells in the
upper respiratory tract and cause cytolysis. FHV-1 also repli-
cates in corneal epithelial cells.46 Although FHV-1 prefers to
replicate in the cooler tissues of the upper respiratory tract,
 CHAPTER 23  Feline Respiratory Viral Infections 241

systemic infection with viremia occurs in some cats infected with

FHV-1 and may be more likely to occur in neonates and debili-
tated cats. FCV and influenza viruses also replicate systemically.
FCV is shed in the urine and feces of infected cats, in addition to
respiratory secretions. Influenza viruses are shed in both respi-
ratory secretions and the feces. Avian origin H5N1 influenza
viruses replicate initially in the lower respiratory tract (type
II pneumocytes and alveolar macrophages) or ­gastrointestinal
tract (for example, after ingestion of an infected bird), which is
followed by severe systemic infection with necrosis and inflam-
mation in multiple organs.38 In contrast, severe systemic infec-
tion is not a feature of H1N1 influenza virus infections in cats.47
Clinical signs of viral URTD occur after an incubation
period of 2 to 6 days, although longer incubation periods are
also possible, and incubation periods as short as 1 to 2 days
can occur in cats infected with influenza viruses.47 Viral shed-
A
ding occurs as early as 24 hours after infection, and often before
the onset of clinical signs. Clinical signs range considerably in
severity from no signs or mild serous ocular discharge to pneu-
monia and death. The most severe signs tend to occur in very
young or elderly debilitated cats. Concurrent immunosuppres-
sive illness or infection with other respiratory pathogens and
opportunistic bacteria also profoundly influences disease sever-
ity. Clinical signs include conjunctivitis, serous or mucopurulent
ocular and nasal discharge and sneezing, and, less commonly,
­cough or tachypnea (Figure 23-2, A). Mucopurulent discharges
result from secondary bacterial infections with opportunistic
pathogens such as Streptococcus spp., Staphylococcus spp., Pas-
teurella multocida, and Escherichia coli. Ocular and nasal dis-
charges may become crusted, and kittens’ eyes may not open as
a result of the sticky exudates; when severe this can be followed
by extensive corneal damage and rupture of the globe. Lethargy, B
inappetence, hypersalivation, and fever may be present in acute FIGURE 23-2  Chemosis, mucopurulent ocular and nasal discharge (A) and lingual
infections. FCV and FHV-1 infections may also lead to pharyn- ulceration (B) in a 6-month-old intact male domestic medium-hair cat with chronic
gitis and laryngitis, which can be accompanied by clinical signs nasal discharge and conjunctivitis. A conjunctival swab specimen tested positive with
of gagging or obstructive respiratory patterns. Ulcerative glos- a PCR assay for FCV RNA and negative for FHV-1, Chlamydia felis, and Mycoplasma
sitis and ulceration of the nasal planum, conjunctiva, and skin spp. DNA. (Courtesy of the University of California, Davis Veterinary Ophthalmology
is more common and severe with FCV infection but can also be Service.)
associated with FHV-1 infection (see Figure 23-2, B). Clinical
features unique to each infection are outlined next.
also cause a severe ulcerative and eosinophilic facial dermatitis
Feline Herpesvirus-1 ­(Figure 23-3). Lesions have also been described elsewhere on the
In neonatal infections caused by FHV-1, damage to upper respi- body in the absence of facial lesions.49
ratory epithelium may lead to osteolysis of the nasal turbinates
and persistent or recurrent sinusitis and rhinitis. Rarely, neuro- Feline Calicivirus
logic signs and reproductive complications such as abortion and FCV infection has been most strongly associated with erosive or
fetal resorption have been observed in infected cats, although ulcerative lesions, which can occur on the nasal planum, tongue,
it is unclear what role FHV-1 itself plays in the pathogenesis lips, and occasionally the conjunctiva and heal over a period of
of these clinical manifestations. FHV-1 is an important cause 2 to 3 weeks.50 Persistent infection with FCV has also been
of corneal disease in cats, and has been implicated as a cause of linked to chronic ulceroproliferative and lymphoplasmacytic
acute and chronic ulcerative (epithelial) keratitis, stromal kera- stomatitis, which involves the mucosa lateral to the palatoglos-
titis, eosinophilic keratitis, corneal sequestra, and uveitis. Stro- sal arches (caudal stomatitis), the alveolar mucosa in the premo-
mal keratitis is thought to be an immunopathologic response lar and molar area, and sometimes the buccal mucosa (alveolar/
to persistent viral antigens. The presence of dendritic corneal buccal mucositis) (Figure 23-4).51-53 Some persistently infected
ulcers is thought to be pathognomonic for FHV-1 infection. cats have isolated hyperemia of the buccal mucosa along the
However, the role that FHV-1 plays as a cause of eosinophilic length of the dental arcade in the absence of significant peri-
keratitis, corneal sequestra, and uveitis requires further study, odontal disease. There is no age predisposition for this condi-
because a clear association between these abnormalities and tion.53 Pyrexia and transient lameness due to synovitis has been
the detection of FHV-1 within corneal tissues has not always described days to weeks after clinical signs of acute FCV infec-
been present.48 Consequences of ocular disease due to FHV-1 tion and after vaccination with certain FCV vaccines. FCV has
include symblepharon (adhesion of an ulcerated conjunctiva to been investigated as a possible cause of feline lower urinary tract
itself or the cornea) and keratoconjunctivitis sicca. FHV-1 can disease (feline interstitial cystitis) and enteritis in cats. Because
242 SECTION 1  Viral Diseases
B superfamily. The lesions that develop are thought to result from
C
FIGURE 23-3  Ulcerative and eosinophilic facial dermatitis secondary to FHV-1
infection. A, 3-year-old female spayed domestic shorthair with a 2-month history of a
crusted lesion on the right dorsal muzzle. B, Fourteen-year-old female spayed domes-
tic shorthair with blepharokeratoconjunctivitis and severe ulcerative facial dermatitis.  ocular discharges, and tachypnea as a result of viral pneumonia,
C, Same cat as in B after 4 months of treatment with famciclovir. Fluorescein stain is pres-
ent. Biopsy in both cats showed severe, diffuse, necrotizing, and eosinophilic dermatitis
with a few intranuclear inclusions bodies. A PCR assay for FHV-1 DNA on a biopsy from the
cat in B was positive. (Courtesy of the University of California, Davis Veterinary Dermatol-
ogy and ­Ophthalmology Services.)
FCV can be shed in the urine and feces of apparently healthy
cats, the significance of the virus in the pathogenesis of these
conditions remains unclear.
Cats with VSD show severe signs of caliciviral URTD,
including anorexia, fever (often >105°F [40.6°C]), weight loss,
FIGURE 23-4  Severe caudal stomatitis in a retrovirus-negative, 8-year-old female
spayed domestic shorthair cat that was evaluated for ptyalism. The cat lived with 12 other
cats. Histopathology revealed severe plasmacytic and neutrophilic inflammation with
multifocal epithelial ulceration and hyperplasia.
oral and footpad ulceration, and nasal and/or ocular discharge.
VSD strains infect not only epithelial cells of the upper respira-
tory tract and oral cavity, but a variety of other cell types, such
as endothelial cells, hepatocytes, pneumocytes, and pancreatic
acinar cells.54 FCV uses feline junctional adhesion molecule
A (JAM-A) as a receptor, a member of the immunoglobulin
disruption of intercellular tight junctions and vasculitis. Distinc-
tive clinical signs of VSD include cutaneous edema, alopecia,
crusting, and ulceration. Edema occurs most commonly on
the head and limbs but may become generalized. Crusting and
ulceration are most prominent on the nose, lips, pinnae, peri-
ocular regions, and distal limbs. Severe respiratory distress due
to pulmonary edema or pleural effusion, or icterus as a result of
hepatic necrosis or pancreatitis, develop in some cats and have
been associated with a poor prognosis. Involvement of the gas-
trointestinal tract, liver, and pancreas may also result in vomit-
ing and/or diarrhea. Cats also develop a coagulopathy, which
can be manifested by petechial and ecchymotic hemorrhages
and, rarely, epistaxis and hematochezia. In peracute infections,
cats die as a result of cardiovascular arrest with few preceding
signs apart from fever.
Influenza Virus Infections
In addition to fever, anorexia, lethargy, conjunctivitis, nasal and
highly pathogenic H5N1 avian origin influenza virus infections
have been associated with neurologic signs such as seizures and
ataxia, which result from nonsuppurative meningoencephalitis
and vasculitis.36 Diarrhea and vomiting have not been observed
in cats infected with H5N1 viruses. Vomiting has been reported
in cats infected with pandemic H1N1 influenza viruses, which
may have a predilection for the gastrointestinal tract.33,34
Physical Examination Findings
Physical examination findings in cats with acute viral URTD
vary from mild serous ocular discharge and conjunctivitis
 CHAPTER 23  Feline Respiratory Viral Infections 243

through to fever, severe mucopurulent ocular and nasal dis-

charges, chemosis, dehydration, thin body condition, stertorous
or stridorous respiration, tachypnea, increased breath sounds
on thoracic auscultation, hypersalivation, and ulceration of the
nasal planum, tongue, and lips. Because ulcerative lesions can
occur at the base of the tongue near the larynx, examination of
the entire tongue should be performed in cats that are febrile
and inappetent, which may require sedation. Cats with VSD
can have edema of the face and lips, icterus, cutaneous ulcer-
ation, evidence of petechial hemorrhages, and abdominal pain.
Ulcerative keratitis with dendritic or geographical corneal ulcer-
ation may be seen in cats with acute FHV-1 infection; chronic
FHV-1 infection may be manifested as stromal keratitis with
neovascularization, pigmentation and fibrosis of the cornea,
symblepharon, and conjunctivalization of the cornea (Figure
23-5). Eosinophilic keratitis manifests as superficial, pink to
A
white vascularized proliferative lesions on the cornea or con-
junctiva; abundant eosinophils are present in smears of corneal
scrapings. Herpetic facial dermatitis is manifested as cutane-
ous ulceration, erythema, exudation, and adherent crusts, most
commonly around the nose and eyes but occasionally on the
trunk and limbs.55 Cats with persistent FCV infection may have
ulceroproliferative caudal stomatitis or buccal/alveolar mucosi-
tis, and exhibit pain on examination of the oral cavity.

Diagnosis
It is not always possible to identify the cause of transmissible
respiratory disease in cats based on clinical signs alone, because
each pathogen produces a similar spectrum of signs. The pres-
ence of corneal ulceration raises suspicion for FHV-1 infection, B
and severe lingual ulceration or facial edema and crusting raises
suspicion for FCV infection, but mixed infections occur and
complicate diagnosis. A history of exposure to other cats pro-
vides support for a diagnosis of viral respiratory disease, but
because FHV-1 infections can recrudesce with stress, potential
or known exposure to other cats is not essential to implicate
respiratory viruses as a cause of disease. Previous immunization
for feline respiratory viruses does not rule out the possibility of
viral URTD. All cats with signs of URTD should have known
retrovirus infection status, because viral URTD is common in
retrovirus-infected cats. When signs such as chronic nasal dis-
charge are present, other etiologies should also be considered,
such as fungal infections, neoplasia, and foreign bodies. Diag-
nosis of eosinophilic keratitis and ulcerative facial dermatitis
may require corneal scrapings or skin biopsy, respectively. C
FIGURE 23-5  A, Ulcerative and stromal keratitis in a 2-year-old male neutered rex
Laboratory Abnormalities cat. FHV-1 infection was suspected. Treatment with famciclovir (375 mg PO q8h), l-lysine
There are no specific CBC, biochemistry profile, or urinalysis (500 mg PO q12h), and cidofovir (0.5%; one drop OD q12h) was associated with clinical
abnormalities that aid in a diagnosis of feline viral respiratory improvement over a 1 month period (B and C). The cat had a history of a corneal seques-
disease. The CBC can be normal or show a mild to moderate trum and subsequently developed eosinophilic keratitis. (Courtesy of the University of
neutrophilia, sometimes with band neutrophils or neutrophil California, Davis Veterinary Ophthalmology Service.)
toxicity. Lymphopenia may be present in severely affected
cats. Serum biochemistry findings in cats with viral respiratory Tracheobronchial lavage specimens from cats with pneu-
disease are usually unremarkable unless disease is severe or monia may show a suppurative or mixed exudate, sometimes
chronic. In cats with VSD, hematologic abnormalities include with evidence of secondary bacterial infection. Aerobic bacterial
mild to severe anemia, thrombocytopenia, neutrophilia, and culture and susceptibility testing and culture for Mycoplasma
lymphopenia. Cats with VSD may also have hypoalbuminemia, spp. are indicated on wash specimens. Organisms such as Pas-
hyperbilirubinemia, mildly increased serum activities of ALT teurella spp., Staphylococcus pseudintermedius, Streptococcus
and AST, and increased serum CK activity. Serum CK activities spp., Escherichia coli, Klebsiella pneumoniae, and some Myco-
up to 11,000 U/L can occur.26 Cats with chronic stomatitis may plasma spp. infect the airways as opportunists. Bacterial culture
have hyperglobulinemia due to a polyclonal gammopathy. of nasal swabs is generally not recommended because it often
244 SECTION 1  Viral Diseases

TABLE 23-2
Diagnostic Assays Available for Respiratory Virus Infections in Cats
Assay Specimen Type Target Performance
Virus isolation Conjunctival, nasal, and caudal Virus Negative results can occur if specimens contain
pharyngeal swabs, transtra- no or low numbers of virus particles. May take
cheal and bronchoalveolar several days and requires specialized techniques
wash specimens, airway and and expertise. May be the most sensitive assay for
lung specimens collected at detection of FCV infections.
necropsy
PCR See Virus isolation Viral nucleic acid Sensitivity and specificity may vary depending on as-
say design. Sensitivity may be low because of brief
shedding for some viruses or low-level shedding in
chronic infections. Assays may not detect all virus
strains. Testing specimens from multiple different
anatomic sites or combining PCR with other diag-
nostic tests can increase sensitivity. Attenuated live
vaccine virus may be detected after vaccination.
Because of subclinical shedding, the significance
of a positive result may be difficult to interpret.
False-negative results may occur as a result of
degradation of viral nucleic acid during specimen
transport.
Serology Serum Antibodies against Interpretation complicated by previous vaccination
respiratory viral and exposure. Acute and convalescent serology
antigens may be useful in outbreak situations that involve a
novel strain of FCV or influenza A virus.
Histopathology Necropsy or biopsy ­specimens FHV-1 inclusions; Detection of FHV-1 inclusions has low sensitivity,
immunostaining and immunostaining and/or PCR is required to
definitively identify the infecting virus.

FCV, Feline calicivirus; FHV-1, feline herpesvirus-1.

leads to growth of normal flora, which has no clinical relevance.
However, resistant Pseudomonas aeruginosa infections can
develop in some cats with chronic URTD that have been treated
repeatedly with antimicrobial drugs.
Diagnostic Imaging
In uncomplicated feline URTD viral infections, plain thoracic
radiographs may be unremarkable or show a mild diffuse
interstitial to bronchointerstitial pattern. Alveolar patterns or
lung lobe consolidation can occur with secondary bacterial
pneumonia.
Microbiologic Tests
Because many cats experience self-limiting disease, attempts to
obtain an etiologic diagnosis should be made when disease per-
sists for longer than 7 to 10 days or is complicated by pneumo-
nia, with lethargy and inappetence. When outbreaks occur in
shelters or the pattern of endemic respiratory disease changes,
attempts to make an etiologic diagnosis are also indicated and
strongly encouraged. Because FCV and FHV-1 can be detected
in apparently healthy cats, it may be difficult to know the sig-
nificance of a positive test result in a single cat with signs of
respiratory disease. In an outbreak situation, collection of mul-
tiple specimen types from several cats with and without clini-
cal signs can facilitate diagnosis and allow interpretation of the
significance of test results. In shelter situations or outbreaks of
severe disease, necropsies can provide valuable information and
should be performed by a veterinary pathologist as soon as pos-
sible after death or euthanasia. Tissues should be submitted for
histopathology (in formalin), bacterial and virus isolation (fresh
tissue), and PCR for respiratory viruses and bacteria (fresh or
frozen tissue; see Chapter 5). There is currently no way to dis-
tinguish FCV strains that cause VSD from other FCV strains.
Assays available to detect feline respiratory viruses include
virus isolation, direct immunofluorescence, and PCR assays
(Table 23-2). Point-of-care ELISA assays developed for use in
humans have been evaluated for detection of H5N1 influenza
virus antigen in cats56 but have low sensitivity and will not be
discussed further. Because influenza viruses and FCV are RNA
viruses that exhibit considerable sequence diversity, virus iso-
lation may offer the greatest sensitivity for detection of these
viruses and enables typing for influenza viruses. In contrast,
FHV-1 loses infectivity more rapidly than FCV and has little
sequence diversity, so PCR is more useful. The use of both
PCR and culture together offers the greatest sensitivity when
it is important to obtain a diagnosis. Serology may be of use
for investigation of outbreaks that involve novel FCV strains or
influenza virus types.
Virus Isolation
FCV, FHV-1, and influenza viruses are readily isolated in cell
culture. Virus isolation for diagnosis of feline respiratory viral

infections is offered to veterinarians by some commercial vet-
erinary diagnostic laboratories that specialize in virology. The
laboratory should be notified if influenza virus infection is a pos-
sibility. Suitable specimens for isolation of respiratory viruses
include conjunctival, nasal, and oropharyngeal swabs, transtra-
cheal or bronchoalveolar lavage specimens, or upper airway tis-
sue or lung obtained at necropsy. Influenza viruses may also be
isolated from feces or rectal swabs. If swabs are used for speci-
men collection, polyester-tipped swabs and specific virus trans-
port media should be used, which are available from commercial
testing laboratories. Cotton swabs should be avoided, because
influenza viruses adhere to the cotton, which may lead to
reduced sensitivity.57 When conjunctival swabs are collected, the
use of topical anesthetics should also be avoided, because they
have the potential to reduce the sensitivity of virus isolation.58
Isolation may take several days. Sensitivity can be very low in
cats with chronic manifestations of infection (for example, more
than 1 week after the onset of clinical signs), because shedding
of respiratory viruses can be transient. In cats with mixed FCV
and FHV-1 infections, FCV may obscure the presence of FHV-1,
because it produces cytopathic effects more rapidly.
Fluorescent Antibody Testing
Fluorescent antibody that specifically detects FHV-1 or FCV
can be applied to conjunctival smears or impression smears
from lung tissue collected at necropsy. The effect of vaccination
with attenuated live vaccines on fluorescent antibody test results
has not been reported. The sensitivity of fluorescent antibody
testing is lower than that of PCR and virus isolation,59 and inex-
perienced laboratory personnel may misinterpret nonspecific
fluorescence as a positive result.
Molecular Diagnosis Using the Polymerase Chain Reaction
Panels of real-time PCR assays that detect feline respiratory
pathogens are offered by some commercial veterinary diagnos-
tic laboratories and are rapid and relatively inexpensive. These
can include assays for FHV-1 and FCV, as well as bacteria such
as Mycoplasma spp., B. bronchiseptica, and C. felis. RT-PCR
assays for the detection of influenza viruses are also available.
The sensitivity and specificity of PCR assays can vary consider-
ably between laboratories. Swabs of the nasal cavity, conjuncti-
val sac, or caudal pharynx; tracheobronchial lavage specimens;
skin biopsies; or upper respiratory tract and lung tissue col-
lected at necropsy are suitable specimens for testing. Topical
anesthetics and fluorescein can reduce the sensitivity of PCR
assays for human herpesviral infections,60 and so they should
be avoided, although one study showed no inhibitory effect
of topical fluorescein or proxymetacaine in cats on FHV-1 or
C. felis PCR results.61 False-negative PCR results can occur in
cats with chronic infections that shed at low levels. Because FCV
and influenza viruses are RNA viruses, false negatives may also
result from degradation of viral RNA during specimen trans-
port or as a result of strain variation.
Positive PCR assay results for FCV and FHV-1 should be
interpreted with caution, because apparently healthy cats can
shed these viruses and, although controversial, FHV-1 DNA
detected in corneal, conjunctival, or nasal biopsy tissue may rep-
resent latent virus. In one shelter, a correlation between the quan-
tity of FHV-1 present in pooled oropharyngeal and conjunctival
swabs as determined using quantitative real-time PCR and the
severity of histologic lesions within the nasal cavity was detected,
with cycle threshold values less than 29 correlating with the most
CHAPTER 23  Feline Respiratory Viral Infections 245
severe histologic lesions.62 In the future, quantitation of viral
nucleic acid within swabs may assist interpretation of the clinical
significance of positive PCR results. PCR can also detect attenu-
ated live vaccine virus that is shed after routine vaccination. How-
ever, this appeared to be an uncommon phenomenon in one study
that used PCR to detect FHV-1 and FCV in 12 cats over a 3-week
period after vaccination with intranasal or parenteral attenuated
live FHV-1 and FCV vaccines. 14 The cats in this study had been
previously exposed to FHV-1, and two had been vaccinated with
FHV-1 and FCV vaccines, which may have reduced shedding
after vaccination. Additional studies are required to determine
the prevalence of positive assay results after vaccination and their
relationship to vaccine virus. In outbreak situations, false-positive
PCR results have the potential to occur if swabs become contami-
nated with virus from the environment or the hands of personnel.
Clean examination gloves should be worn for each cat, and the
swab should touch only the anatomic site to be tested.
Serologic Diagnosis
Serologic assays (such as ELISA or serum neutralization assays)
that detect antibodies to FHV-1 and/or FCV are offered on a
commercial basis to veterinarians. Unfortunately, serology is
not useful for diagnosis, because of vaccine titer interference
and the high prevalence of subclinical exposure to these viruses
in the cat population. Titers to FCV may vary depending on the
degree of homology between the infecting virus and the FCV
strain used in the assay. Nevertheless, serology has been useful
to investigate some outbreaks of VSD. Serology has also been
used to predict protection from infection, although cats with
negative titers can still have some degree of immunity, and cats
with positive titers may develop illness after challenge.
Serologic assays for influenza virus exposure are based
on serum neutralization or hemagglutination-inhibition (see
Chapter 2) and are specific for the influenza virus strain of inter-
est (i.e., H5N1, H1N1).
Pathologic Findings
Gross lesions in cats with viral URTD include cutaneous and lin-
gual ulcerations, evidence of keratitis in cats infected with FHV-
1, submandibular or retropharyngeal lymph node enlargement,
mucosal hyperemia, and mucopurulent exudate within the nasal
cavity and trachea. In cats with pneumonia, the lungs may be
edematous with diffuse or multifocal consolidated areas. Histo-
pathology may show fibrinosuppurative and necrotizing stomati-
tis, rhinitis, tracheitis, and/or alveolitis. When FHV-1 is present,
intranuclear viral inclusions may be found within epithelial cells
of the upper respiratory tract (Figure 23-6). Osteolytic changes
within the nasal turbinates may also be identified in cats infected
with FHV-1. Virus can be detected in epithelial cells using immu-
nohistochemistry or fluorescent antibody staining. Skin biopsies
from cats with ulcerative herpesviral dermatitis show epidermal
ulceration and necrosis that extends into the superficial dermis,
and infiltration with degenerated neutrophils, eosinophils, and
fewer numbers of histiocytes, plasma cells, and lymphocytes. In
some skin biopsies, intranuclear viral inclusions are identified.55
Necropsy findings in cats with VSD are variable. Frequently
reported findings include individual hepatocellular necrosis and
dissociation with minimal inflammation, acute interstitial pneu-
monia, and free pleural and abdominal fluid.26-28,31 Acute nec-
rotizing vasculitis of subcutaneous and oral submucosal vessels
has been described.32 Intestinal crypt necrosis and pancreatitis
have been reported in experimentally infected cats.
246 SECTION 1  Viral Diseases
A 1 mm
B tage of requiring only twice daily administration.
FIGURE 23-6  A, Cross-section of the nares of a shelter cat with rhinitis due to infec-
tion by FHV-1. Ulceration is most severe on the right side. Hematoxylin and eosin stain.
B, Higher magnification of the nasal epithelium that shows rhinitis and the presence of
intraepithelial, intranuclear inclusion bodies. (From Burns RE, Wagner DC, Leutenegger
CM, et al. Histologic and molecular correlation in shelter cats with acute upper respiratory
infection. J Clin Microbiol 2011;49(7):2454-2460. Images courtesy Dr. Patricia Pesavento,
University of California, Davis Veterinary Anatomic Pathology Service.)
In addition to acute respiratory lesions, cats infected with
H5N1 influenza viruses can have widespread necrosis and
inflammation in many organs.
Treatment and Prognosis
Acute Upper Respiratory Disease
Supportive care is the mainstay of treatment for acute feline
URTD and includes subcutaneous fluid therapy, antimicrobial
drugs for secondary bacterial infections, and enteral nutrition.
Appetite stimulants such as diazepam may be useful in some
cats, but if inappetence persists for longer than 3 days, feeding
tube placement is indicated. Severely affected cats may require
hospitalization in isolation and treatment with parenteral fluids,
nebulization, and supplemental oxygen. Acute signs generally
resolve within 2 to 3 weeks with supportive care, but some cats
experience frequent disease relapses and chronic complications
such as keratitis and persistent stomatitis. Cats with VSD may
require treatment with colloids. Mortality rates that exceed
50% can occur in outbreaks of VSD.31
Herpetic Keratoconjunctivitis and Facial
Dermatitis
The use of antiviral drugs should be considered for cats with
severe and recurrent or persistent manifestations of FHV-1
infection such as keratitis, severe conjunctivitis, and ulcerative
facial dermatitis. Antiviral drugs are discussed in more depth
in Chapter 7 (see Table 7-2 for dosages). Referral to a veteri-
nary specialist for assessment of an underlying cause should
be considered for cats with signs of chronic URTD. Acyclovir
and its prodrug, valacyclovir, cause unacceptable toxicity when
administered systemically to cats, and so their use is not recom-
mended. In contrast, oral famciclovir is well tolerated by adult
cats and kittens and results in significant clinical improvement
and decreased viral shedding in cats with acute and chronic her-
pesviral disease (see Figure 23-3, B and C).63,64 The prodrug of
famciclovir, penciclovir, also has potential to be a useful drug
for treatment of feline herpesviral infections. Because FHV-1
infection can deplete conjunctival goblet cells,64a frequent appli-
cation of a topical mucinomimetic that contains hyaluronic acid
is recommended in conjunction with famciclovir.
Topical ophthalmic preparations that contain idoxuridine,
trifluridine, vidarabine, or acyclovir can be used to treat herpes-
viral keratitis, although the true efficacy of these drugs has not
been well studied. Frequent topical application is required (5 to
6 times daily), and prolonged use can cause corneal irritation or
ulceration. Idoxuridine and vidarabine are better tolerated by
cats than trifluridine; vidarabine is effective against idoxuridine-
resistant strains. Cidofovir and ganciclovir show greater prom-
ise as topical treatments and are highly active against FHV-1
in  vitro. Topical cidofovir decreases disease severity and viral
shedding in experimentally infected cats,65 and has the advan-
Human recombinant IFN-α and recombinant feline IFN-ω
inhibit FHV-1 replication in vitro and have been administered
parenterally, topically (for keratitis), and orally to cats with
FHV-1 infection. Obvious clinical responses to treatment have
not been uniformly observed, but controlled clinical trials that
involve large numbers of cats are lacking. Improvement was
noted when IFN-α was given subcutaneously 2 days before
inoculation of cats with FHV-1,66 but the effect of parenteral
IFN-α after the onset of clinical signs requires further investiga-
tion. Parenteral administration may be followed by the develop-
ment of neutralizing antibodies beyond 3 weeks of treatment.
Lysine has shown efficacy for treatment of herpesviral con-
junctivitis in cats when administered as tablets67 and can reduce
reactivated shedding by latently infected cats.68 However, when
administered to shelter cats, there was no reduction in URTD,
possibly because of the stress of tablet administration.69 Dietary
formulation with lysine has also not been effective for manage-
ment of URTD in shelters and in fact may contribute to a wors-
ened outcome.70 Administration of lysine tablets or paste could
be considered for client-owned cats with chronic herpesviral
disease but is not recommended in shelter situations.
The use of glucocorticoids to treat refractory keratitis is con-
troversial, because it may reactivate virus and worsen disease.
However, eosinophilic keratitis may require treatment with top-
ical glucocorticoids. Because the role of FHV-1 in this condition
is unclear, topical antiviral medications such as cidofovir are
often given concurrently. Topical glucocorticoids are contrain-
dicated when corneal ulceration is present, so the eyes should
first be carefully examined after staining with fluorescein. Cats

with ulcerative eosinophilic keratitis may require ­systemic glu-
cocorticoid treatment until the ulcers heal. Referral to a veteri-
nary ophthalmologist is recommended.
Chronic Gingivostomatitis
Caudal stomatitis in cats infected with FCV is often refractory
to medical treatment. Clinical cure occurs in 50% to 60% of
cats and there is significant improvement in 30% to 40% of cats
after 1) extraction of teeth in the vicinity of lesions, 2) treat-
ment with antimicrobial drugs with activity against anaerobic
and gram-positive aerobic bacteria, and 3) the use of antiseptic
mouthwashes (see Chapter 21). Cats that fail to respond to this
treatment may require long-term treatment with antimicrobial
and antiinflammatory drugs, although one study showed mini-
mal improvement in cats with refractory stomatitis that were
treated with prednisolone.53 Recombinant feline IFN-ω has also
been used subcutaneously and orally to treat caudal stomati-
tis. In a randomized, multicenter, controlled, double-blinded
study of cats with refractory caudal stomatitis and alveolar/
buccal mucositis, oromucosal treatment with rfIFN-ω (0.1 mil-
lion units q24h) was associated with significant improvement in
lesions and decreased pain scores from day 0 to day 90.53
Influenza Virus Infections
Oseltamivir has been used to treat captive wild and domestic cats
with influenza virus infections,33,71 but it was not effective. The
optimal dose of this drug in cats is not known. Therefore, cats with
suspected influenza virus infections should be treated supportively.
Immunity and Vaccination
Immunity to respiratory viruses depends on cell-mediated and
systemic and local (mucosal IgA) humoral immune responses.
Maternal antibody to respiratory viruses can persist until 14
weeks of age,15 although other kittens may be susceptible to
infection at 6 weeks of age.
Vaccines for FHV-1 and FCV have been available for decades,
but these do not provide complete protection, and disease con-
tinues to be widespread in the cat population. Infection of kittens
before completion of the primary vaccine series contributes to this
problem. Both inactivated and attenuated live FHV-1 and FCV
vaccines are available, and attenuated live vaccines are available
for parenteral or intranasal administration. Inactivated vaccines
contain adjuvant, and their use should be reserved for immu-
nosuppressed cats (such as retrovirus-infected cats) or pregnant
cats, if deemed necessary. When infections are a problem in kit-
tens in breeding catteries, vaccination of the queen before mating
is preferable to vaccination during pregnancy and may prolong
the duration of maternal antibody persistence.50 Vaccination
as early as 4 weeks of age (but not before) could be considered
in catteries with high rates of viral URTD. The primary series
should consist of vaccines administered every 3 to 4 weeks of age
until no earlier than 16 weeks of age.
No respiratory viral vaccine prevents infection, the carrier
state, or reactivation of FHV-1 infection with stress, but they
can reduce disease severity. Whether a cat contracts feline viral
URTD after vaccination depends on factors such as virus strain
virulence, the challenge dose, environmental temperature and
humidity, and the presence of underlying immunosuppressive
disease or other concurrent disease.
For FHV-1 infections, vaccination can reduce the duration of
shedding, viral infectivity (presumably as a result of complexing
CHAPTER 23  Feline Respiratory Viral Infections 247
of virus by antibody), and the load of latent virus in the trigemi-
nal ganglia.15,72,73 It is unclear if the frequency of reactivation is
reduced, although this is the case for herpesviral vaccines in other
species (such as the shingles vaccine for humans). The intranasal
FHV-1 vaccine virus can itself become latent, but whether par-
enteral FHV-1 vaccine virus becomes latent is unknown. Most
FCV vaccines do not prevent shedding, and virus shedding from
the oropharynx can actually increase after FCV vaccination,
because of shedding of attenuated live vaccine virus.
Because FHV-1 strains are antigenically similar, the immune
response to one FHV-1 strain cross protects against other FHV-1
strains. Greater variability in cross protection occurs for FCV
strains. Vaccines that contain F9, the most widely used FCV vac-
cine strain, provide partial protection against aerosol challenge
with heterologous strains.74 Cross-neutralization studies have
shown that the antigenic composition of current field FCV strains
may differ enough from the original F9 strain that the level of
cross-protection induced by F9 vaccines may be suboptimal. As
a result, it has been suggested that new FCV strains should be
incorporated into FCV vaccines. However, vaccines that con-
tain F9 still appear to generate cross-reactive antibody to a large
number of currently circulating strains.75 A nonadjuvanted, inac-
tivated vaccine that contains two FCV strains (G1 and 431) is
available in Europe, which provided greater reduction in clini-
cal scores after challenge with a heterologous strain that the use
of either strain alone and also reduced shedding.76 Exposure of
vaccinated cats to field strains of FCV may also serve to boost
immune responses and ultimately broaden protection. Cats that
are not exposed to field strains, such as isolated indoor cats, may
be more susceptible to severe disease when hypervirulent strains
appear. An adjuvanted, inactivated vaccine for VSD that contains
a single hypervirulent strain was introduced in the United States
in 2007. However, the degree to which this vaccine cross-protects
against other hypervirulent strains is unknown, and outbreaks
cease when infection control measures are implemented. Because
of this and the increased risk of sarcoma formation with adju-
vanted vaccines, the usefulness of the vaccine has been questioned.
Parenteral vaccines that contain FCV and FHV-1 are gener-
ally safe, although mild signs of URTD have the potential to
occur if the cat licks the site of vaccination or aerosolization
of vaccine occurs during administration. Intranasal vaccines
are less well tolerated by cats and can be occasionally be fol-
lowed by mild to moderate postvaccinal signs of URTD. This
may be problematic in shelter environments where clinical
signs of URTD warrant the need for separate housing. On the
other hand, intranasal vaccines are advantageous in this situa-
tion because of rapid onset of immunity, with partial protection
after 2 days and significant protection after 4 days.15 They can
also overcome maternal antibody at a young age, although some
injectable vaccines may also still be effective at an early age.
Because vaccine virus is propagated in feline kidney cells, there
has been concern that parenteral administration of feline vac-
cines might lead to chronic renal inflammation when immune
responses to vaccine components cross-react with endogenous
renal antigens.77 Further studies are required to determine
whether this is clinically important.
Vaccine boosters are recommended 1 year after completion
of the primary series, and at 3-yearly intervals thereafter in cats
with low levels of exposure to respiratory viruses. For cats in
contaminated environments such as catteries, boarding facili-
ties, or large multicat households with frequent introductions,
annual vaccination may be more appropriate. The duration of
248 SECTION 1  Viral Diseases
immunity after vaccination remains incompletely understood.
Although partial protection persists for several years after vac-
cination,78 the degree of protection does wane with time, at
least for cats that receive no natural boostering in the interim
(i.e., from exposure to field virus). In one report, relative FHV-1
and FCV vaccine efficacies were 95% and 85%, respectively,
when challenge occurred a few weeks after primary vaccination
with an inactivated vaccine.79 This compared with 52% and
63%, respectively, when challenge occurred after 7.5 years.80
In another study, a 50% reduction in clinical scores occurred
after challenge 1 year after administration of an attenuated live
FHV-1 vaccine, compared with around 75% after challenge
4 weeks after vaccination.81 Similar results were observed for
an inactivated FCV vaccine. In the field, natural boostering as
a result of contact with virus shed by other cats or subclinical
reactivation may maintain immunity for longer periods of time.
Prevention
In addition to vaccination, reduction of stress and overcrowding
is critical for prevention of URTD, especially that due to FHV-
1. Proper hand washing and disinfection in shelters, boarding
facilities, and veterinary hospitals to prevent fomite transmis-
sion is also very important, and dedicated food and water bowls
should be used. Inactivation of FCV requires the use of disin-
fectants such as sodium hypochlorite (household bleach diluted
1:30), potassium peroxymonosulfate (Trifectant, Virkon S), or
accelerated hydrogen peroxide. Veterinarians should remember
that all cats, whether apparently healthy or sick, can be a source
CASE EXAMPLE
Signalment: “Boris”, a 7-year-old male neutered Burmese cat
from northern California.
History: Boris was brought to the University of California, Davis,
Veterinary Medical Teaching Hospital for a 3-day history
of inappetence, head-shaking, gagging, hypersalivation,
coughing, and sneezing. The gagging occurred throughout
the day and occasionally was followed by vomiting. The
vomit consisted of bile-stained fluid. Boris had not been
drinking. He was taken to a local emergency clinic where
bilateral serous nasal discharge was noted, and he was
treated for 24 hours with intravenous crystalloid fluids and
amoxicillin. Blood work showed hypokalemia (4.0 mEq/L,
reference range 4.5 to 5.3 mEq/L), hyperglycemia (292 mg/
dL, reference range 70 to 150 mg/dL), neutrophilia (12,920
cells/μL, reference range 2500 to 12,500) and lymphopenia
(136, reference range 1500 to 7000). FeLV and FIV serology
was negative. A urinalysis showed a specific gravity of
1.050, pH of 7, and 1+ proteinuria and glucosuria. Lateral
and dorsoventral radiographs of the thorax and abdomen
showed moderate to severe gas distention of the proximal
esophagus and stomach. Profuse salivation and gagging
continued, and he was referred to a specialty clinic because
of concerns for a possible gastrointestinal foreign body.
There had been no diarrhea, no history of travel or trauma,
and no recent surgical procedures. A new indoor plant was
brought into the house 2 days before the onset of clinical
of FHV-1 or FCV infection, and cats examined for routine vac-
cination purposes may be susceptible to nosocomial infection
with these viruses. Cats should be housed individually (unless
they are from the same household and tolerate close proximity
to one another). Barriers between cats should be impermeable,
and cats with URTD should be separated by at least 4 to 5 feet
to prevent aerosol transmission. If possible, cats that enter large
multicat environments and shelters should be quarantined for
3 weeks to identify cats that are incubating disease. When all
other control measures fail in breeding catteries, early weaning
and isolation of kittens from 4 weeks could be considered.
In regions where highly pathogenic H5N1 avian influenza
virus infections have been identified in birds, cats should be kept
indoors. Restriction zones defined for this purpose consist of an
area within a 10-km radius of an outbreak of influenza in birds
for 30 days after the discovery of infected birds.36 Cats should
also not be fed raw poultry. Separation of cats from avian spe-
cies in shelters and hospital situations should be maintained.
Public Health Aspects
FHV-1 and FCV do not infect humans. Although cats can
acquire influenza virus infections from humans, the risk of
transmission from cats back to humans is unknown. Neverthe-
less, cats suspected to have influenza virus infections (such as
during a recognized outbreak) should be isolated, the handling
of these cats should be minimized, and appropriate protective
attire should be worn, which should ideally include properly
fitted high-density surgical masks.
signs. He was last immunized for FHV-1, FCV, FPV, and rabies
1 year previously. Boris was housed completely indoors and
was exposed to one other cat that only occasionally had
access to the outdoors. His diet consisted of a commercial
dry cat food.
Physical Examination:
Body Weight: 4.8 kg
General: Lethargic, but responsive, 5% to 7% dehydrated. Held
head extended and gagged and hypersalivated throughout
the examination; seemed unable to swallow. Vomited bile-
stained fluid once during the examination. T = 100.8°F (38.2°C),
HR >240 beats/min, respiratory rate = 24 breaths/min, mucous
membranes pink, CRT = 1 s. The cat’s haircoat was unkempt.
Eyes, Ears, Nose, and Throat: Bilateral serous nasal discharge
was present. A linear ulcer was identified on the right lateral
aspect of the tongue.
Musculoskeletal: Body condition score 4/9. Ambulatory.
Cardiovascular and Respiratory: Other than tachycardia, no
other abnormalities were detected.
Gastrointestinal and Genitourinary: Mild abdominal
distention was present.
Lymph Nodes: All lymph nodes were < 1 cm in diameter.
Laboratory Findings:
CBC:
HCT 34.2% (30%-50%)
MCV 51.0 fL (42-53 fL)
MCHC 32.5 g/dL (30-33.5 g/dL)
WBC 12,600 cells/µL (4500-14,000 cells/µL)
Neutrophils 11,844 cells/µL (2000-9000 cells/µL)

Band neutrophils 126 cells/µL
Lymphocytes 126 cells/µL (1000-7000 cells/µL)
Monocytes 252 cells/µL (50-600 cells/µL)
Eosinophils 126 cells/µL (150-1100 cells/µL)
Platelets 989,000 platelets/µL (180,000-500,000 platelets/
µL). The neutrophils showed slight toxicity, and there
were a few Heinz bodies
Serum Chemistry Profile:
Sodium 141 mmol/L (151-158 mmol/L)
Potassium 4.0 mmol/L (3.6-4.9 mmol/L)
Chloride 108 mmol/L (117-126 mmol/L)
Bicarbonate 27 mmol/L (15-21 mmol/L)
Phosphorus 4.5 mg/dL (3.2-6.3 mg/dL)
Calcium 8.1 mg/dL (9.0-10.9 mg/dl)
BUN 21 mg/dL (18-33 mg/dL)
Creatinine 1.1 mg/dL (1.1-2.2 mg/dL)
Glucose 212 mg/dL (63-118 mg/dL)
Total protein 5.1 g/dL (6.6-8.4 g/dL)
Albumin 2.2 g/dL (2.2-4.6 g/dL)
Globulin 2.9 g/dL (2.8-5.4 g/dL)
ALT 83 U/L (27-101 U/L)
AST 50 U/L (17-58 U/L)
ALP 16 U/L (14-71 U/L)
Cholesterol 147 mg/dL (89-258 mg/dL)
Total bilirubin 0 mg/dL (0-0.2 mg/dL)
Endoscopy: Boris was anesthetized and an oral examination
performed, which showed only mild dorsal pharyngeal
lymphoid hyperplasia. Rhinoscopy showed a moderate
amount of mucus and mild diffuse erythema of the nasal
mucosa. Gastroduodenoscopy revealed focal areas of gastric
erosion with hemorrhage.
Treatment: Boris was treated with IV crystalloid fluids (LRS
with 20 mEq/L KCl at 20 mL/h), ampicillin (23 mg/kg IV
q8h), enrofloxacin (2.6 mg/kg IV q24h), sucralfate (0.25 g PO
q8h), and famotidine (0.25 mg/kg IV q12h). He continued to
hypersalivate, and he vomited approximately 100 mL of fluid
SUGGESTED READINGS
Ali A, Daniels JB, Zhang Y, et  al. Pandemic and seasonal human
influenza virus infections in domestic cats: Prevalence, association
with respiratory disease, and seasonality pattern. J Clin Microbiol.
2011;49:4101-4105.
Gaskell R, Dawson S, Radford A, et  al. Feline herpesvirus. Vet Res.
2007;38:337-354.
Marschall J, Hartmann K. Avian influenza A H5N1 infections in cats.
J Feline Med Surg. 2008;10:359-365.
Radford AD, Coyne KP, Dawson S, et  al. Feline calicivirus. Vet Res.
2007;38:319-335.
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CHAPTER 23  Feline Respiratory Viral Infections 249
twice daily. After the first day of hospitalization, the owners
reported that their other cat had developed signs of URTD,
with sneezing and nasal discharge. Enteral or parenteral
nutrition was discussed with the owner, but on the evening
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Necropsy Examination: Gross necropsy examination
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Comments: This cat died of a severe and overwhelming
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CHAPTER 24
Feline Poxvirus Infections
Malcolm Bennett
Overview of Feline Poxvirus Infections
First Described: Feline poxvirus infection was first described
in 19781
Causes: Poxviruses (family Poxviridae). Cowpox virus infec-
tion is by far the most frequently reported poxviral infec-
tion of both cats and dogs. Other poxvirus infections,
including parapoxvirus (assumed to be orf virus) and
raccoonpox virus infections, have also been described
in pets. Other poxviruses are also likely to infect cats and
dogs but go unreported.
Affected Hosts: Cats, less often dogs, and a variety of other
mammalian host species
Geographic Distribution: Cowpox virus is limited to Europe
and Asia. Infections with other poxviruses have been
described in North America.
Mode of Transmission: Entry through a break in the skin
Major Clinical Signs: Single or multiple, papular, and crusted
skin lesions. Fever, lethargy, inappetence, and signs of
pneumonia can also occur in affected cats.
Differential Diagnoses: Allergic dermatitis, facial dermatitis
caused by feline herpesvirus-1, nocardiosis, mycobacte-
riosis, cutaneous lymphoma, autoimmune dermatoses
such as pemphigus foliaceus, cutaneous drug reactions
Human Health Significance: Many poxviruses, including cow-
pox and orf viruses, are zoonotic, and owners of affected
animals need to be advised of the threat to their health.
Etiology and Epidemiology
Cowpox Virus
Cowpox virus is a member of the Orthopoxvirus genus within
the family Poxviridae, which are large, enveloped, double-
stranded DNA viruses.2 Other orthopoxviruses include the
viruses of smallpox (now eradicated), vaccinia (the smallpox
vaccine), monkeypox (which is endemic to central Africa but
was recently introduced to, and eradicated from, North Amer-
ica), and a range of other mammalian orthopoxviruses. All
orthopoxviruses are closely related genetically and antigenically;
therefore, vaccinia virus can be used to immunize against all
orthopoxvirus infections. Cowpox virus has the largest genome
of all the orthopoxviruses. Different strains of cowpox virus
exist, as determined by both biologic characteristics and nucleo-
tide sequence. Whether these merely reflect geographic variation
of the virus (and therefore its main hosts and pathogenicity) or
252
a range of independent virus species is still to be determined.
For the purpose of this account, however, they are regarded as
a single viral species.
Despite its name, cowpox is rarely described in cattle; cow-
pox virus circulates primarily in wild rodents. It has a wide
host range that includes not only rodents, cats, dogs, cattle,
and human beings, but a variety of captive wildlife mam-
mals. The main route of transmission among rodents is not
known, although, as for many orthopoxviruses, the respira-
tory route is probably important. In most accidental hosts,
which include domestic animals and human beings, the most
frequent source of infection is through a break in the skin.
This gives rise to the “primary” lesion. The main reservoirs of
cowpox virus are voles and, to a slightly lesser extent, Apode-
mus mice. As these rodent species undergo annual population
cycles, breeding mainly in the summer and fall months, viral
transmission and prevalence has strong seasonality. Transmis-
sion to accidental hosts such as cats, dogs, people, and zoo
animals therefore also occurs mainly (but not exclusively) in
the late summer, fall, and early winter. Cats probably become
infected directly from rodent hunting, whereas infection in
other species is often acquired from a “liaison” or “amplify-
ing” host. These are accidental hosts in which the virus rep-
licates to higher titers than in the reservoir rodent hosts; thus
they bridge the epidemiologic gap between the reservoir and
the host of interest. The main liaison hosts for human cowpox
are the domestic cat (and, in the past, possibly cattle), fol-
lowed by peridomestic rodent species such as rats. Rats can
also be a source of infection for captive wildlife species. The
virus is very hardy and survives in scabs for weeks or months,
including in the environment, where it may be a source of
human infection.
Raccoonpox Virus
Raccoonpox virus is also an orthopoxvirus. Remarkably little
is known about its epidemiology, or indeed about the other
known North American orthopoxviruses, skunkpox, and
volepox viruses, which together form a distinct clade. Raccoon-
pox virus appears to be fairly host specific but was isolated from
a cat with cowpox-like disease in Canada.3
Raccoonpox virus was originally isolated from the lungs of
apparently healthy raccoons in the northeastern United States,
and serologic surveys in the same area suggested that more than
20% of wild raccoons had antibody to the virus. Experimen-
tal infection of raccoons also caused no obvious disease.4 The
narrow host range, low zoonotic and pathogenic potential, and
endemicity of raccoonpox virus to North America has led to
its development as a vector for recombinant vaccines for use in
American wildlife species.

Parapoxviruses
The parapoxviruses comprise a different genus from the
orthopoxviruses in the family Poxviridae. Parapoxviruses are
genetically very similar to each other (but significantly differ-
ent antigenically and genetically from the orthopoxviruses), and
differentiation of individual species is difficult.5 Parapoxvirus
infections have been described in cats in several countries, and
when characterized, the causative virus has been shown to be
orf virus.6 Orf occurs worldwide, mainly in sheep and goats,
and is readily transmissible to human beings.
Parapoxviruses, like many other poxviruses, survive in the
environment for long periods—possibly months or even years
under the right conditions. So although cats can become infected
through direct contact with sheep or goats, it is more likely that
infection occurs through contact with a contaminated fomite—
perhaps a piece of barbed wire or fencing that contacts a preex-
isting wound. The rarity of parapoxvirus infection in cats makes
its study very difficult.
Clinical Features
Signs and Their Pathogenesis
Cowpox Virus
In many hosts, such as human beings, dogs, and cattle, the pri-
mary lesion of cowpox virus infection, plus perhaps some virus
replication in draining lymph nodes, may be as far as disease
progresses. In cats and many captive wild mammalian species,
however, the virus replicates in monocytes and macrophages
and spreads to multiple organs, particularly the lungs and the
spleen. Here, the virus replicates further, giving rise to a second-
ary viremia, which is often associated with pyrexia and mild or
occasionally severe systemic signs. Pneumonia can itself be fatal,
but usually the secondary viremia leads to a “secondary rash”
of lesions distributed across the skin and mucosae.
The signs of cowpox vary according to the host species. The
classical presentation in cats is of a primary skin lesion, often with
secondary bacterial infection, usually on a forelimb or the face,
thought to arise from the inoculation of virus into a wound—
perhaps a bite wound—while hunting infected wild rodents. In
some cases, severe necrotizing facial dermatitis develops. The pri-
mary lesion may be followed by lethargy and possibly pyrexia for
up to 10 days, after which multifocal skin lesions appear. These
first develop as papules (Figure 24-1), which become ulcers up to
1-cm in diameter that crust over and heal over 4 to 6 weeks. This
leaves small patches of alopecia until the hair regrows. In cats
such as Siamese, the hair can grow back a darker color, and scars
are often visible for years in cats if their hair is clipped. Infection
of dogs is less common, and in dogs, lesions are usually more
localized and the systemic disease much less severe.6-8
In many captive wild Felidae, pneumonia is more com-
mon than in domestic cats, and mortality is high. Similarly,
elephants and rhinoceroses in central European zoos develop
severe systemic disease and often die—indeed, this can be such
a severe disease that many European zoological parks vaccinate
their elephants. There are few reports of fatal cowpox in cap-
tive primates,9 but there are anecdotal reports of high rates of
subclinical seroconversion in other primate populations. Other
affected zoo-kept species include giant anteater, beavers, okapi,
banded mongoose, jaguarondi, and the cavy. In wild rodent
reservoir hosts, clinical signs are absent or, at least, subtle.
Population-level studies of naturally infected voles and mice
show that infection may be associated with decreased fecundity
CHAPTER 24  Feline Poxvirus Infections 253
FIGURE 24-1  Secondary papules and crusted skin lesions in a cat with ­cowpox virus
infection.
and decreased survival.10 When lesions are seen in rodents, they
are often in “accidental” rodent hosts such as rats.
Other Poxviruses
Raccoonpox has only been reported once in a domestic cat,3 in
which it caused a disease that resembled the primary lesion of
cowpox. The cat was initially evaluated for a swollen, crusty
lesion on a thoracic limb digit, and 4 weeks later infection of the
neighboring digit developed. Widespread secondary lesions did
not develop, and the cat recovered completely. Human infection
with a raccoonpox recombinant vaccine led to a single lesion
on the inoculated finger, which became swollen and painful
and was accompanied by axillary lymphadenopathy. Complete
recovery occurred within 4 weeks.
Parapoxvirus infections have been described in cats in the
United Kingdom and New Zealand.5,10 The British cat devel-
oped multiple crusted lesions on its face and dorsum that healed
within a few weeks. Two cats from farms in New Zealand had
more severe clinical signs. One had a recurrent lesion on the
paw of a pelvic limb. The other had a nonhealing lesion on the
paw of a thoracic limb that progressed, despite surgery, over
four months. Both cats were euthanized.
Diagnosis
Definitive diagnosis of poxvirus infections requires laboratory
identification of the causative virus. In Europe, where cowpox
virus is endemic, clinical signs combined with identification of
an orthopoxvirus probably suffice to make a diagnosis. Else-
where, however, more extensive laboratory tests are required to
identify the causative virus more precisely.
Microbiologic Tests
Diagnostic assays available for poxvirus infections are shown
in Table 24-1.
Cell Culture
The best specimens to collect for virus isolation are fresh skin
lesions, although virus can be found in crusts from healing
lesions for several weeks.11 No special transport media are
254 SECTION 1  Viral Diseases

TABLE 24-1
Diagnostic Assays Available for Poxvirus Infections
Assay Specimen Type Target Performance
Virus isolation Skin biopsies, crusts Poxviruses May not be widely available in some regions. Sensitive for di-
agnosis of orthopoxviruses, but parapoxviruses are difficult
to culture.
Polymerase chain Skin biopsies, crusts Poxvirus DNA Sensitivity and specificity may vary depending on assay design;
reaction (PCR) assays may not differentiate between orthopoxviruses.
Availability on a commercial basis may be limited in some
regions. Assays for parapoxviruses are not widely available.
Histopathology Skin biopsies, nec- Poxvirus inclusions Histopathology is similar for cowpox virus and raccoonpox
ropsy specimens virus. Confirmation of poxvirus infection requires immuno-
histochemistry or PCR.
Electron micros- Skin crusts Virus particles May not be widely available, turnaround time can be slow,
copy and may be expensive. Orthopoxviruses and parapoxviruses
can be differentiated based on their morphology.

needed. Lesions can be placed into a dry container for shipping

to the laboratory at room temperature. The virus replicates in
a variety of cell lines, in which it causes an obvious cytopathic
effect (CPE) that resembles that of feline herpesvirus-1. Unlike
most feline viruses, however, cowpox virus grows easily in cell
lines derived from other mammalian species, including primates.
Lesion material is ground or finely chopped and freeze-thawed
several times to release virus particles. CPE usually appears
within 24 to 76 hours and comprises plaques of syncytia fol-
lowed by rounding and loss of cells from the center of the
plaque. Staining with hematoxylin and eosin reveals large, intra-
cytoplasmic, eosinophilic inclusion bodies. Further confirmation
can be by immunostaining (e.g., immunofluorescence) to iden-
tify the virus as an orthopoxvirus, or PCR assay and sequencing.
Although raccoonpox virus grows in a variety of cell lines,
diagnosis is more likely to be made by histopathology and PCR
assays. This is because the virus is unlikely to be suspected ini-
tially, and biopsy and histology would be the first response of
most veterinarians. Parapoxviruses are notoriously difficult to
grow in cell culture, requiring primary cell cultures.

Molecular Diagnosis Using the Polymerase Chain Reaction

Some diagnostic laboratories offer PCR assays for diagnosis
of poxvirus infection, which can be both sensitive and, if com-
bined with sequencing, specific. Orthopoxviruses are so similar
genetically that PCRs targeted at many orthopoxvirus genes
only identify orthopoxviruses to the genus level. PCRs that dif-
ferentiate between orthopoxvirus species have been described,
but such assays tend to be available through research or other
specialist laboratories. Of course, in much of Europe, cowpox
virus is the only orthopoxvirus known to infect cats and dogs, so
identification to the species level is often not necessary. Unlike
orthopoxviruses, for which worries over bioterrorism have led
to the development of several sensitive and specific PCR assays,
molecular assays for parapoxviruses are not widely available.
Direct Electron Microscopy
Electron microscopy of skin lesions is a more rapid approach
to diagnosis than cell culture and is fairly sensitive. Nega-
tive-stained suspensions of skin lesion material allow typical
FIGURE 24-2  Electron micrograph of a cowpox virus particle. In contrast, parapoxvi-
ruses have the appearance of a new ball of yarn, with threadlike surface tubules arranged
in a crisscross fashion.
orthopoxvirus particles to be seen in most cases (Figure 24-2).
The diagnosis of parapoxvirus infection usually relies on elec-
tron microscopy and visualization of the characteristic parapox-
virus morphology, which resembles a “ball of yarn.”
Serologic Diagnosis
Previous cowpox virus infection can be diagnosed by various
serologic assays including immunofluorescent antibody, ELISA,
and virus neutralization assays. The prevalence of orthopoxvi-
rus antibodies in domestic cats in Europe is usually only 1% to
2%, so a consistent clinical history combined with an antibody
titer is reasonably specific.

FIGURE 24-3  Histopathology of a feline poxvirus lesion that shows epithelial hyper-
plasia and large numbers of eosinophilic intracytoplasmic inclusion bodies.
Pathologic Findings
Many cases of cowpox in cats, dogs, and humans are diagnosed
by histopathology.6,12,13 The histology of cowpox is quite char-
acteristic; the virus causes hyperplasia and hypertrophy of the
epithelium, with infected cells often containing eosinophilic,
intracytoplasmic inclusions that can be readily observed in
routine hematoxylin and eosin–stained sections (Figure 24-3).
These A-type inclusions comprise virus-encoded protein that
forms a protective mass around newly formed virus particles.
As the epithelium thickens, cells in the middle layers lyse, which
gives rise to fluid-filled lacunae that, when large enough, can
be seen with the naked eye as the vesicles (or pocks/pox) from
which these viruses get their name. Usually the vesicles quickly
ulcerate in cats. Older lesions undergo secondary bacterial
infection and subsequent neutrophil infiltration before heal-
ing. Immunostaining is useful in histopathologic diagnosis,
especially in older lesions or those with secondary bacterial
infection.
The histopathology of raccoonpox resembles that of
cowpox.3 Hyperplastic, hypertrophic epithelium is present
with eosinophilic, intracytoplasmic A-type inclusion bodies.
Because North American orthopoxviruses are quite distinct
from “old world” (Eurasian) orthopoxviruses, PCR assays and
sequencing can readily characterize the virus, even from fixed
material.11
Histologic examination of parapoxvirus lesions from the
New Zealand cats revealed epithelial hyperplasia giving rise to
multiple papillary projections from the skin surface, covered in
a thick layer of compacted keratin.5 Orf virus does not pro-
duce the A-type inclusions often seen with orthopoxviruses, but
occasional basophilic or amphophilic inclusion bodies may be
present (so-called B-type inclusions, which consist of condensed
areas of virus replication).
CHAPTER 24  Feline Poxvirus Infections 255
Treatment and Prognosis
Treatment of poxvirus infections is supportive. There are no spe-
cific antiviral agents available. Most cats with cowpox recover
completely, but some, particularly if immunosuppressed by
concurrent infection or treatment with, for example, glucocorti-
coids, develop larger, persistent skin lesions, and/or pneumonia.
Cats that develop pneumonia often die despite supportive care.
Prevention
Housing cats indoors should help to prevent poxvirus infection
in cats. No vaccine is available. However, canarypox-vectored
vaccines are used to prevent rabies virus and FeLV infection in
cats and distemper in dogs.
Public Health Aspects
Cowpox virus infection in humans is usually limited to a skin
lesion at the site of inoculation (often on the hand or face)
accompanied by local lymphadenopathy and flu-like symp-
toms. The lesions can be painful, and an important differential
diagnosis is cutaneous anthrax. In Europe, most human cases
can be traced to contact with an infected cat; infection from
rodents or dogs is a much less frequent event. Although most
patients recover uneventfully, immunosuppression can lead to
more extensive lesions and, rarely, even death.12,14-16 Although
thought not to be zoonotic, a recombinant raccoonpox vaccine
caused lesions in a laboratory worker.17 Orf is readily transmis-
sible to humans, usually from contact with sheep and goats.
Although transmission from an infected cat to a human being
has not been reported, human orf associated with a cat scratch
was reported from Utah in the United States.13
SUGGESTED READINGS
Fairley RA, Whelan EM, Pesavento PA, et al. Recurrent localised cuta-
neous parapoxvirus infection in three cats. N Z Vet J. 2008;56:
196-201.
Glatz M, Richter S, Ginter-Hanselmayer G, et al. Human cowpox in a
veterinary student. Lancet Infect Dis. 2010;10:288.
Yager JA, Hutchison L, Barrett JW. Raccoonpox in a Canadian cat. Vet
Dermatol. 2006;17:443-448.
REFERENCES
1. Thomsett LR, Baxby D, Denham EM. Cowpox in the domestic cat.
Vet Rec. 1978;103:567.
2. Bennett M, Smith GL, Baxby D. Cowpox virus. In: Mahy BWJ,
Regenmortel MHVV, eds. Encyclopedia of Virology. 3rd ed.
Oxford: Academic Press; 2008.
3. Yager JA, Hutchison L, Barrett JW. Raccoonpox in a Canadian cat.
Vet Dermatol. 2006;17:443-448.
4. Thomas EK, Palmer EL, Obijeski JF, et al. Further characterization
of Raccoonpox virus. Arch Virol. 1975;49:217-227.
5. Fairley RA, Whelan EM, Pesavento PA, et al. Recurrent localised
cutaneous parapoxvirus infection in three cats. N Z Vet J.
2008;56:196-201.
6. Bennett M, Gaskell CJ, Gaskell RM, et al. Poxvirus infection in the
domestic cat: some clinical and epidemiological observations. Vet
Rec. 1986;118:387-390.
7. Kaysser P, von Bomhard W, Dobrzykowski L, et al. Genetic diver-
sity of feline cowpox virus, Germany 2000-2008. Vet Microbiol.
2010;141:282-288.
8. von Bomhard W, Mauldin EA, Breuer W, et al. Localized cowpox infec-
tion in a 5-month-old Rottweiler. Vet Dermatol. 2011;22:111-114.
256 SECTION 1  Viral Diseases
9. Girling SJ, Pizzi R, Cox A, et al. Fatal cowpox virus infection in
two squirrel monkeys (Saimiri sciureus). Vet Rec. 2011;169:156.
10. Hamblet CN. Parapoxvirus in a cat. Vet Rec. 1993;132:144.
11. Bennett M, Baxby D, Gaskell RM, et al. The laboratory diagnosis
of Orthopoxvirus infection in the domestic cat. J Small Anim Pract.
1985;26:653-661.
12. Baxby D, Bennett M, Getty B. Human cowpox 1969-93: a review
based on 54 cases. Br J Dermatol. 1994;131:598-607.
13. Frandsen J, Enslow M, Bowen AR. Orf parapoxvirus infection
from a cat scratch. Dermatol Online J. 2011;17:9.
14. Baxby D, Bennett M. Poxvirus zoonoses. J Med Microbiol.
1997;46:17-20:28-33.
15. Favier AL, Flusin O, Lepreux S, et al. Necrotic ulcerated lesion in
a young boy caused by cowpox virus infection. Case Rep Dermatol.
2011;3:186-194.
16. Glatz M, Richter S, Ginter-Hanselmayer G, et al. Human cowpox
in a veterinary student. Lancet Infect Dis. 2010;10:288.
17. Rocke TE, Dein FJ, Fuchsberger M, et al. Limited infection upon
human exposure to a recombinant raccoon pox vaccine vector.
Vaccine. 2004;22:2757-2760.
CHAPTER 25
Pseudorabies
Jane E. Sykes and Sarah D. Cramer
Overview of Pseudorabies
First Described: 1902 (Hungary, Aujeszky)1
Cause: Suid herpesvirus-1 (Family Herpesviridae, subfamily
Alphaherpesvirinae, genus Varicellovirus)
Affected Hosts: Domestic and feral swine, cattle, horses,
sheep, goats, dogs, cats, bears, raccoons, foxes, hedge-
hogs, opossums, deer, Florida panther, jackals, coyotes,
nonhuman primates (macaques, marmosets), chickens,
pigeons, geese, ducks, buzzards, sparrow hawks, rabbits,
guinea pigs, rats, and mice
Geographic Distribution: Worldwide
Mode of Transmission: Most often ingestion of uncooked
pork by-products or pig carcasses
Major Clinical Signs (Dogs): Variable, but may include fever,
lethargy, hypersalivation, muscle stiffness, variable facial
pruritus, ataxia, behavioral changes, vomiting, diarrhea,
and/or respiratory distress
Differential Diagnoses: Rabies, canine distemper, toxicoses
(e.g., organophosphates, heavy metals, ethylene glycol,
strychnine)
Human Health Significance: Humans appear to be resistant
to infection with suid herpesvirus-1.
Etiology and Epidemiology
Pseudorabies (Aujeszky’s disease) is a notifiable disease caused
by the alphaherpesvirus suid herpesvirus-1 (SuHV-1). Swine
are the natural hosts for SuHV-1, but other domestic and wild
mammals, including ruminants, dogs, cats, raccoons, rabbits,
and rodents, are susceptible to infection and fatal encephalitis.
The name pseudorabies was applied because the clinical signs
caused by the virus in rabbits resemble those of rabies. SuHV-1
has been eradicated from domestic swine through use of vacci-
nation programs in many countries, including Finland, Sweden,
Norway, the Netherlands, Denmark, Austria, Switzerland, Ger-
many, Hungary, Slovakia, the United Kingdom, Canada, the
United States, and New Zealand. The virus continues to circulate
among wild boar and feral swine, which act as a reservoir for the
virus.2 Australia is also free of pseudorabies. Aujeszky’s disease
is a widespread problem in domestic swine herds in Asia, Latin
America, and some parts of Europe.2
SuHV-1 is shed in the saliva and nasal discharges of swine,
and transmission can occur by direct contact with these dis-
charges or aerosols, or ingestion of infected swine carcasses or
other infected animals, especially wildlife. The virus can survive
on fomites and in carcasses for several weeks. In swine, clini-
cal signs depend on the age at exposure and virus strain. Sub-
clinical infections and shedding occur commonly. When older
pigs do develop signs, they typically develop respiratory disease.
Encephalitis occurs in piglets or in adult pigs that are infected
with virulent strains. Reproductive disease and fetal loss can
occur in pregnant sows. Recovered pigs develop latent infection
of neuronal tissues with reactivation and shedding of virus in
semen and nasal, oral, or vaginal secretions following stress.
Disease in dogs is uncommonly reported. However, clusters
of affected dogs have been described over the past decade, pri-
marily in hunting dogs that contact wild swine from parts of
Europe (France, Germany, Belgium) and the southern United
States (Florida and Oklahoma).2-7 This has followed global
expansion of wild swine populations. Disease in dogs may be
more likely to occur in regions that are densely populated with
wild swine, such as the southern United States (Figure 25-1)
and parts of Europe such as Germany, France, Spain, Poland,
and the Czech Republic.2 Dogs that reside on swine farms may
also become infected.8 In some reports, packs of dogs have been
infected after consumption of uncooked offal from infected pigs
or pig carcasses.9,10 Dogs of any age, sex, and breed can develop
disease, but dog-to-dog transmission does not occur. Cats can
be infected after ingestion of pork waste or infected rodents,
but reports of naturally occurring Aujeszky’s disease in cats are
extremely rare.11 Because subclinical infections and nonspecific
signs are common in swine, identification of pseudorabies in
dogs may be the first indication that disease is present in a swine
herd or wild swine population.
Clinical Features
Signs and Their Pathogenesis
After inhalation or ingestion, SuHV-1 replicates in the orophar-
ynx and is taken up by sensory nerve endings. It then travels
by retrograde axonal transport to the sensory nerve ganglia
and then to the central nervous system (CNS), which results
in ganglioneuritis and encephalitis, especially within the brain-
stem. Infection can also lead to ganglioneuritis of the cardiac
autonomic plexuses with associated myocardial degeneration,12
as well as degeneration of intestinal myenteric ganglia.10 Clini-
cal signs appear after a short incubation period that typically
ranges from 1 to 6 days, but incubation periods up to 10 days
have been reported.9 The duration of signs ranges from 6 to
96 hours.13 The most commonly reported sign is hypersaliva-
tion. This occurs in most affected dogs, although it may not be
present early in the course of illness.10,13,14 Other signs include
lethargy, fever, dysphagia, gastrointestinal signs, muscle stiff-
ness, ataxia, head-pressing, vestibular signs (circling, head tilt,
257
258 SECTION 1  Viral Diseases

FIGURE 25-1  Geographic distribution of feral swine in the United States. (From
Southeastern Cooperative Wildlife Disease Study, College of Veterinary Medicine, The Uni- FIGURE 25-2  Evidence of facial self-mutilation in a 2-year-old intact male Cata-
versity of Georgia. http://128.192.20.53/nfsms. Last accessed May 2, 2012.) houla hunting dog with pseudorabies. Severe exudative dermatitis is present over the
right eye, external ear canal and muzzle. (Reprinted from Cramer SD, Campbell GA, Njaa
BL, et  al. Pseudorabies virus infection in Oklahoma hunting dogs. J Vet Diagn Invest
2011;23:915-923.)
BOX 25-1
Clinical Signs in 25 Dogs with Pseudorabies from atrioventricular dissociation.12 Although facial pruritus is con-
the United States sidered a classic clinical sign, in one case series, it was present in
only 18% of dogs.14 Death occurs within 96 hours (and usually
Ptyalism: 100% 48 hours) after the onset of clinical signs. Sudden death may
Anorexia: 84% occur, possibly as a result of acute myocarditis.9,14
Ataxia: 76%
Wandering: 64% Physical Examination Findings
Tachypnea: 64% Physical examination findings in dogs with pseudorabies include
Dyspnea: 60% mental obtundation, hypersalivation, dehydration, muscle stiff-
Vocalization: 56% ness, tachypnea or respiratory difficulty, and facial pruritus.
Pruritus: 52% Cutaneous excoriations and edema due to self-trauma may be
Neck stiffness: 48% identified (Figure 25-2). Other neurologic signs include ataxia,
Vomiting: 36% head tilt, circling, hyperesthesia, behavioral abnormalities,
Muscle spasms: 36% anisocoria, mydriasis, nystagmus, slow or absent pupillary light
Aggression: 36% responses, recumbency, and coma. Fever may be present early in
Trismus: 28% the course of infection (up to 108°F or 42°C),13 but terminally,
Dysphagia: 24% hypothermia can occur. A variety of cardiac arrhythmias may
Abnormal pupillary light responses: 20% also be identified.
Seizures: 16%
Mydriasis: 12% Diagnosis
Monroe WE. Clinical signs associated with pseudorabies in dogs. J Am Diagnosis of pseudorabies in dogs and cats is based on a com-
Vet Med Assoc 1989;195:599-602. patible history (i.e., exposure to domestic or wild swine in
regions where the virus is present), suggestive clinical signs,
and histopathology and/or virus detection methods such as
nystagmus), recumbency, intense pruritus and self-mutilation, immunofluorescent antibody staining, immunohistochemis-
vocalization, behavior changes such as pacing and aggression, try, virus isolation, or PCR assay on tissues collected at nec-
and tachypnea or respiratory distress (Box 25-1). Uncom- ropsy (Table 25-1). Antemortem diagnosis of pseudorabies in
monly, apparent blindness, facial paralysis, ptosis, photopho- dogs and cats using virus detection methods has not yet been
bia, abnormal facial sensation, and lacrimation occur. Pruritus described.
generally occurs on the face but can also occur on the shoulder
and forelimbs.10,13 The pruritus may result from infection of Laboratory Abnormalities
neurons that innervate those regions.15 Gastrointestinal signs Laboratory abnormalities in dogs with pseudorabies are non-
are common and include vomiting and diarrhea, which may be specific. Mild anemia and moderate leukocytosis due to a neu-
hemorrhagic (e.g., hematemesis or melena).6 Seizures and coma trophilia may be present. Affected hunting dogs have also had
may also occur. A variety of cardiac arrhythmias have been eosinophilia, hyperglobulinemia, hypoalbuminemia, and hyper-
reported in experimentally infected dogs, which include ventric- glycemia.7 In these dogs, concurrent gastrointestinal parasitism
ular premature complexes, ventricular tachycardia, sinus arrest, may have explained the eosinophilia, hypoalbuminemia, and
atrial tachycardia, atrial fibrillation, wandering pacemaker, hyperglobulinemia. Analysis of the CSF may reveal increased
second-degree heart block, T- and P-wave abnormalities, and CSF protein concentration and mononuclear pleocytosis.
 CHAPTER 25  Pseudorabies 259

TABLE 25-1
Diagnostic Assays Available for Pseudorabies in Dogs and Cats
Assay Specimen Type Target Performance
Histopathology Necropsy specimens, especially Meningoencephalitis with eo- Necropsy diagnosis
brainstem or ganglia sinophilic intranuclear inclusions;
SuHV-1 antigen with IHC, IFA or
DNA with in situ hybridization
PCR CNS, tonsils, possibly ­biopsies SuHV-1 DNA Unknown whether PCR could be
of self-traumatized skin or used for antemortem diagnosis.
tonsillar swabs Not widely available.
Virus isolation CNS, tonsils, occasionally other SuHV-1 Specialized procedure. False negatives
tissues such as skin ­biopsies or ­occur commonly.
lung tissue

CNS, Central nervous system; IFA, immunofluorescent antibody; IHC, immunohistochemistry; SuHV-1, suid herpesvirus-1.

Microbiologic Tests
Serologic Diagnosis
Although serology using a variety of antibody detection meth-
ods (such as ELISA and virus neutralization) is used to identify
exposure in swine herds, it has not been useful for antemor-
tem diagnosis in dogs and cats. This likely reflects the rapid
course of disease and insufficient time before death for antibody
responses to develop.

Virus Isolation
Although not always successful, SuHV-1 can be isolated from
tissues (such as the CNS and tonsils) in pig kidney or Crandell-
Rees feline kidney cell monolayers. Immunofluorescent anti-
body can then be used to confirm the presence of the virus in
cells. The sensitivity of virus isolation as an antemortem test
(e.g., on saliva or oropharyngeal swabs) is not known. Haired
skin from regions of pruritus may also be suitable.
FIGURE 25-3  Histopathology in pseudorabies encephalitis. Trigeminal ganglion of
Molecular Diagnosis Using the Polymerase Chain Reaction the dog in Figure 25-2. Neutrophilic, lymphocytic, and histiocytic infiltrates are present.
Conventional and real-time PCR assays have been described Ganglion cells are admixed with necrotic cellular debris, and neuronophagia is prominent.
Within a single ganglion cell is an equivocal intracytoplasmic inclusion (arrow). Bar = 50 µm.
for detection of SuHV-1 in brain tissue collected at necropsy.7
(From Cramer SD, Campbell GA, Njaa BL, et al. Pseudorabies virus infection in Oklahoma
Whether PCR assays are useful for antemortem diagnosis is not hunting dogs. J Vet Diagn Invest 2011;23:915-923.)
known, but PCR could be attempted on biopsies of pruritic
regions or tonsillar swab specimens.

Pathologic Findings Treatment and Prognosis

Abnormalities seen on gross postmortem examination in dogs
with pseudorabies include facial excoriations, tonsillar enlarge-
ment, gastrointestinal mucosal hyperemia and ulceration,
myocardial hemorrhage, and pulmonary edema and conges-
tion. On histopathologic examination, perivascular infiltrates
of lymphocytes, neutrophils, and macrophages may be present
in the brainstem and medulla oblongata, with neuronophagia,
neuronal degeneration and necrosis, focal gliosis, and eosino-
philic intranuclear inclusion bodies within neurons and astro-
cytes (Figure 25-3). Involvement of the spinal cord was reported
in a cat.11 Other changes in dogs include suppurative tonsillar
inflammation with necrosis, myocardial degeneration, fibri-
noid vasculitis, and pulmonary edema. Immunohistochemistry,
immunofluorescence, or in situ hybridization can be used to
identify virus within neurons.8
Other than supportive care, there is no known treatment for
pseudorabies in dogs and cats. Disease is almost always rapidly
progressive and fatal.
Immunity and Vaccination
Although vaccines are used in swine to reduce losses where the
disease is enzootic, vaccines are not available for dogs and cats,
because of the sporadic incidence of the disease.
Prevention
Pseudorabies can be prevented by cooking pork by-products
fed to dogs and cats, preventing contact between infected swine
260 SECTION 1  Viral Diseases
and dogs in regions where the disease is enzootic, and possibly
through control of rodents.
Public Health Aspects
Humans appear to be refractory to infection with suid
herpesvirus-1.
SUGGESTED READINGS
Cramer SD, Campbell GA, Njaa BL, et al. Pseudorabies virus infection
in Oklahoma hunting dogs. J Vet Diagn Invest. 2011;23:915-923.
Muller T, Hahn EC, Tottewitz F, et al. Pseudorabies virus in wild swine:
a global perspective. Arch Virol. 2011;156:1691-1705.
REFERENCES
1. Aujeszky A. Über eine neue Infektienkrankheit bei Haustieren.
Zentralbl Bakteriol I Orig. 1902;32:353-357.
2. Muller T, Hahn EC, Tottewitz F, et al. Pseudorabies virus in wild
swine: a global perspective. Arch Virol. 2011;156:1691-1705.
3. Thaller D, Bilek A, Revilla-Fernandez S, et  al. Diagnosis of
Aujeszky’s disease in a dog in Austria. Wien Tierarztl Monatsschr.
2006;93:62-67.
4. Cay AB, Letellier C. Isolation of Aujeszky’s disease virus from two
hunting dogs in Belgium after hunting wild boars. Vlaam Dierge-
neeskd Tijdschr. 2009;78:194-195.
5. Toma B, Dufour B. Transmission de la maladie d’Aujeszky
des sangliers aux suides domestiques. Epidemiol Sante Anim.
2004;45:115-119.
6. Buergelt CD, Romero CH, Chrisman CL. Pseudorabies in two
dogs. Vet Med. 2000;95:439-442.
7. Cramer SD, Campbell GA, Njaa BL, et  al. Pseudorabies virus
infection in Oklahoma hunting dogs. J Vet Diagn Invest.
2011;23:915-923.
8. Quiroga MI, Nieto JM, Sur J, et al. Diagnosis of Aujeszky’s disease
virus infection in dogs by use of immunohistochemistry and in-situ
hybridization. Zentralbl Veterinarmed A. 1998;45:75-81.
9. Hugoson G, Rockborn G. On the occurrence of pseudorabies in
Sweden. II. An outbreak in dogs caused by feeding abattoir offal.
Zentralbl Veterinarmed B. 1972;19:641-645.
10. Gore R, Osborne AD, Darke PG, et al. Aujeszky’s disease in a pack
of hounds. Vet Rec. 1977;101:93-95.
11. Hara M, Shimizu T, Nemoto S, et al. A natural case of Aujeszky’s
disease in the cat in Japan. J Vet Med Sci. 1991;53:947-949.
12. Olson GR, Miller LD. Studies on the pathogenesis of heart
lesions in dogs infected with pseudorabies virus. Can J Vet Res.
1986;50:245-250.
13. Monroe WE. Clinical signs associated with pseudorabies in dogs.
J Am Vet Med Assoc. 1989;195:599-602.
14. Hawkins BA, Olson GR. Clinical signs of pseudorabies in the dog
and cat: a review of 40 cases. Iowa State Univ Vet. 1985;47:116-119.
15. Takahashi H, Yoshikawa Y, Kai C, et al. Mechanism of pruritus
and peracute death in mice induced by pseudorabies virus (PRV)
infection. J Vet Med Sci. 1993;55:913-920.
CHAPTER 26
Viral Papillomatosis
Jane E. Sykes and Jennifer A. Luff
Overview of Papillomatosis in Dogs and Cats
First Described: The transmissibility of canine papilloma-
viruses was described in the late 1800s; electron micro-
scopic descriptions occurred in the late 1960s1,2
Cause: Papillomaviruses (family Papillomaviridae)
Affected Hosts: A variety of animal species, but papillomavi-
rus types are relatively species specific
Geographic Distribution: Worldwide
Mode of Transmission: Cutaneous or mucosal inoculation
through trauma
Major Clinical Signs: Exophytic or endophytic papillomas,
pigmented plaques, in situ or invasive squamous cell
carcinomas
Human Health Significance: Canine and feline papillomavi-
rus types do not appear to infect humans, although some
feline papillomavirus types resemble some human papil-
lomavirus types
Etiology and Epidemiology
Papillomaviruses cause warts, or papillomas, in a variety of ani-
mal species (Figure 26-1). They are non-enveloped, icosahedral
viruses with a circular double-stranded DNA genome. Papillo-
maviruses have also been associated with malignant transfor-
mations within the skin and mucous membranes (Box 26-1).3 In
humans, certain papillomavirus types are more likely than oth-
ers to induce malignant transformation. Papillomavirus DNA
has been detected using PCR assays on the skin of dogs and cats
and in the oral cavity of dogs with no signs of papillomas4-6; it
is not clear whether this represents subclinical infection or just
carriage of papillomaviruses.7 Because of this, the association
of papillomaviruses with disease in dogs and cats, especially
squamous cell carcinomas (SCCs), remains somewhat unclear.
Papillomaviruses are resistant in the environment and survive
detergents and high temperatures. Exposure to papillomaviruses
is widespread in the dog population based on serologic studies.8
Congenital or acquired deficiencies in cell-mediated immunity
may predispose to papilloma formation.9-13
Papillomaviruses are relatively host species specific. The num-
ber of different papillomavirus types identified in dogs and cats
has expanded dramatically in recent years as a result of the use
of molecular techniques. Correlations appear to exist between
papillomavirus types and clinical manifestations and progression
of disease, although host immune status is also important. Papil-
lomaviruses are classified on the basis of the L1 capsid protein
gene sequence. At least nine types occur in dogs; an additional
five novel types have been described that are not fully classified
(Table 26-1).14-21 Canis familiaris papillomavirus (canine papil-
lomavirus 1) is associated with oral papillomas and rarely with
cutaneous endophytic (inverted) papillomas and invasive cutane-
ous SCCs.10,22,23 Other canine papillomavirus types have been
associated with cutaneous inverted and exophytic papillomas,
cutaneous pigmented plaques, or cutaneous in situ SCCs. Papil-
lomavirus DNA has been detected in a few canine oral SCCs.23,24
However, most oral and cutaneous SCCs in dogs are negative
for papillomavirus DNA. The eight canine papillomavirus types
have been allocated to three different genera, Lambda, Tau, and
Chi.7,17 Chi papillomaviruses have been associated with cutane-
ous plaque formation. Cocker spaniels and Kerry blue terriers
may be predisposed to cutaneous papillomas, and pug dogs are
predisposed to pigmented plaques. Breed predispositions to papil-
lomavirus infections may reflect the presence of congenital defects
in cell-mediated immunity; however, this is not yet proven.
Papillomavirus infections are uncommonly reported in cats
when compared with dogs. They have been associated with
plaque-like lesions (hereafter referred to as feline plaques), dys-
plastic skin lesions, multicentric Bowenoid in situ SCCs (BISC),
cutaneous invasive SCCs, and feline sarcoids (also known as
fibropapillomas). The complete viral genome sequence for
two feline papillomaviruses types has been described, as well
as several stretches of additional novel feline papillomavirus
sequences. Feline sarcoids have been associated with feline sar-
coid-associated papillomavirus (FeSarPV) infection, for which
cattle may be a reservoir.25 Sarcoids have been described in cats
from North America, New Zealand, the United Kingdom, Swe-
den, and Australia, and affected cats are often young (<5 years),
male, outdoor cats from rural environments. Felis domes-
ticus papillomavirus 1 (FdPV-1) has been detected in feline
viral plaques.26,27 Felis domesticus papillomavirus 2 (FdPV-2)
FIGURE 26-1  Structure of a papillomavirus. The virus is non-enveloped and has a
icosahedral capsid composed of two different capsid proteins (L1 and L2).
261
262 SECTION 1  Viral Diseases
BOX 26-1
Spectrum of Clinical Manifestations Associated with
Papillomavirus Infections in Dogs and Cats
Dogs
Oral papillomatosis
Cutaneous exophytic papillomas (warts)
Cutaneous endophytic papillomas (inverted warts)
Pigmented cutaneous plaques
Cutaneous in situ SCCs*
Invasive cutaneous SCCs
Oral SCCs
Cats
Feline viral plaques
Bowenoid in situ carcinomas
Invasive cutaneous SCCs
Feline sarcoids
*SCCs, squamous cell carcinomas. The majority of SCCs in dogs and
cats have not been associated with papillomavirus infection.
and other papillomaviruses, including papillomaviruses with
homology to human papillomaviruses, have been detected in
cats with plaques, BISCs, and invasive SCCs.27-31 FdPV-2 is
strongly associated with feline plaque formation.32 The finding
of papillomavirus DNA with homology to the DNA of human
papillomaviruses in cats suggests the possibility of transmission
of papillomaviruses between humans and cats; however, addi-
tional studies are needed.30,31Although papillomavirus DNA
has been detected in a feline oral SCC,33 papillomaviruses do
not seem to be responsible for most oral SCCs in cats.34
Clinical Features
Signs and Their Pathogenesis
Infection with papillomaviruses follows inoculation of the virus
through microabrasions in the skin caused by physical trauma.
Initial infection and viral amplification occurs within keratino-
cytes of the stratum basale.35 As basal keratinocytes undergo
differentiation, early viral proteins (E proteins) are expressed
and associate with regulators of the cell cycle to stimulate cell
cycle progression.35 This leads to enhanced proliferation of
cells in the stratum spinosum and stratum granulosum, with
accumulation of infected cells and papilloma formation (Figure
26-2). With the exception of feline sarcoids, which remain as
latent (or nonproductive) infections, late viral products, includ-
ing the virus capsid proteins (L1 and L2), are first expressed
in cells of the stratum spinosum. Papillomaviruses are nonlytic
viruses, and mature virions are shed with cells that exfoliate
from the stratum corneum or the nonkeratinized cells of the
mucous membranes. Infected cells develop a characteristic cyto-
pathic effect, which consists of cytoplasmic vacuolization. Tens
of thousands of virus particles are produced by each infected
cell. Malignant transformation occurs as a result of the effect of
certain viral products (also known as oncoproteins) on host cel-
lular signals that are responsible for regulation of the cell cycle
and/or apoptosis.36 For example, some papillomavirus types
produce proteins that degrade p53 and retinoblastoma pro-
tein (RB), which are important tumor suppressors.37 In human
TABLE 26-1
Spectrum of Papillomavirus Types and Disease
Associations in Dogs
CPV Type* Genus Clinical Manifestation
1 Lambda Oral papillomas, cutaneous endo-
phytic papillomas, invasive SCCs
2 Tau Cutaneous endophytic and exophytic
papillomas (warts)
3 Chi Cutaneous pigmented plaques, in
situ and invasive SCCs
4, 5 Chi Cutaneous pigmented plaques
6 Lambda Endophytic papillomas
7 Tau Exophytic papillomas and in situ
SCC
8 Chi Cutaneous pigmented plaques
9 Cutaneous pigmented plaques
*CPV, Canine papillomavirus. Five additional types associated with
cutaneous pigmented plaques exist that have not yet been definitively
classified at the time of writing. (Luff JA, Affolter VK, Yeargan B, et al.
Detection of six novel papillomavirus sequences in canine pigmented
plaques. J Vet Diagn Invest 2012;24[3]:576-580.)
papillomavirus infections, accidental integration of the human
papillomavirus (HPV) genome can occur into host cell DNA,
although this does not seem to be necessary for oncogenesis.36
Oral papillomas most commonly occur in young dogs and
often regress spontaneously over a 4- to 6-week period.7 They
can also occur as a result of immunosuppression, such as after
treatment with cyclosporin, less commonly chronic glucocor-
ticoid treatment, or chronic infection with Ehrlichia canis.
Rarely, papillomas in the caudal oropharynx obstruct respi-
ration (Figure 26-3). Cutaneous pigmented plaques, to which
pugs and miniature schnauzers are predisposed, are generally
not of clinical significance but in some situations can progress
to SCCs.19,38,39 Cutaneous exophytic and endophytic papillo-
mas usually occur as solitary masses.7 Exophytic papillomas are
proliferative nodules, whereas endophytic papillomas are cup-
shaped lesions with a central, keratin-filled pore (Figure 26-4).
Feline plaques are flat, often pigmented lesions that resemble
canine papillomas histologically. Feline sarcoids are tumors that
resemble equine sarcoids. Multicentric BISCs appear as mul-
tiple, hyperpigmented plaquelike lesions.
Physical Examination
Canine Oral Papillomatosis
Oral papillomas can occur on the lips, mucocutaneous junctions,
tongue, pharynx, eyelids, and infrequently haired skin7 (see Fig-
ure 26-3; Figure 26-5). They may be accompanied by halitosis,
ptyalism, and oral discomfort. Early lesions are smooth and
raised, but as they enlarge they may become cauliflower-like.
They may be pigmented or nonpigmented.
Canine Cutaneous Exophytic Papillomas
Cutaneous exophytic papillomas are wartlike projections that
appear anywhere on the body, but especially the lower limbs
and footpads.
 CHAPTER 26  Viral Papillomatosis 263

Dead keratinocyte
releasing viral particles
Dead keratinocyte
Keratin
SC
1 Keratohyaline
granule
SG
Nucleus
Koilocyte
Cytoplasmic
vacuolization
SS
Inclusion
2 body
SB

Basal lamina B
4

A 3
FIGURE 26-2  Schematic representation of the events of papillomavirus infection in the epidermis. A, (1) The primary infection occurs in a cell of the stratum basale (SB) after the virus gains
entry via a microabrasion. The virus replicates and produces only early proteins, which leads to increased cellular proliferation. (2) Progeny of the initial infected cells migrate laterally and move
upwards, where they accumulate in the stratum spinosum (SS) and stratum granulosum (SG). (3) Cellular differentiation ultimately occurs, and large numbers of virions are produced in association
with papilloma development. Virions are shed with exfoliating keratinocytes of the stratum corneum (SC). (4) Detail from 3. B, Immunoperoxidase staining of papillomavirus antigen in a cutane-
ous plaque from a dog. (B courtesy of Luff J, University of California, Davis. In MacLachlan NJ, Dubovi EJ, eds. Fenner’s Veterinary Virology, 4 ed. Burlington, MA: Academic Press [Elsevier]; 2011.)

Canine Endophytic Papillomas Diagnosis

Endophytic (or inverted) papillomas are cup-shaped cutaneous
nodules that range in size from 2 mm to 2 cm in diameter with a
central depression (see Figure 26-4). Several different variations
have been described, but they are rare and poorly understood.7
Single or multiple lesions can occur anywhere on the body. They
have also been described in the interdigital space.9
Canine Pigmented Plaques
Pigmented plaques usually range in size from 1 mm to 1 cm,
but in one dog progressed to larger sizes (up to 8 cm). Multiple
dark, hyperkeratotic flat lesions are typically present on the ven-
trum or medial aspects of the limbs.39
Squamous Cell Carcinomas
SCCs are often ulcerated, nodular masses that may be found
alongside pigmented plaques or cutaneous papillomas when
malignant transformation occurs (Figure 26-6).
Feline Sarcoids
Feline sarcoids are solitary, firm, ulcerated, or nonulcerated
nodules that occur most commonly on the nasal philtrum,
nares, upper lip, digits, or tail tip, but have also been reported at
other locations such as the eyelids and pinnae (Figure 26-7).25,40
Feline Plaques and BISC
In cats, plaques are usually a few millimeters in diameter or lin-
ear lesions. They are usually pigmented and occur on the head,
neck, dorsal thorax, or abdomen. They can be ulcerated.41 It
may be difficult to differentiate plaques from BISCs, which also
appear as pigmented plaques with crusting and ulceration. The
two may occur together simultaneously.
The history and gross appearance of oral papillomas in dogs
is generally sufficient for diagnosis. For other papillomas and
plaques, the diagnosis can be confirmed with biopsy and histo-
pathology. A full-thickness biopsy that includes adjacent nor-
mal skin should be collected. The presence of papillomaviruses
can be identified using PCR assays, immunohistochemistry, in
situ hybridization, or electron microscopy, although these tech-
niques are primarily used on a research basis. Caution should be
used when interpreting the results of PCR assays because papil-
lomaviruses can be detected on normal skin, so results should
be interpreted in association with clinical signs. Identification of
virus within lesions using immunohistochemistry, in situ hybrid-
ization, or electron microscopy is more supportive of causality.
Microbiologic Tests
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays can be used to detect papillomaviruses within
biopsy or cytobrush specimens from dogs and cats. Rolling
circle amplification has been used on fresh biopsy specimens
to amplify whole circular genomic DNA from papillomavirus
lesions.7
Pathologic Findings
Histopathology of canine exophytic papillomas generally
reveals epidermal hyperplasia (acanthosis), extensive hyper-
keratosis, and clumped keratohyalin granules in the stratum
spinosum. The term koilocytosis is used to describe cells with
papillomavirus-induced cytopathic effects, which consist of
extensive cytoplasmic vacuolation and nuclear pyknosis. Intra-
nuclear inclusion bodies may also be present; these occur in the
264 SECTION 1  Viral Diseases

FIGURE 26-4  Inverted papilloma on the abdomen of a flat-coated retriever.

(From Lange CE, Favrot C. Canine papillomaviruses. Vet Clin North Am Small Anim Pract
2011;41:1183-1195.)

C B
FIGURE 26-3  A, Oral papillomas in the pharyngeal region of a 5-year-old male neu- FIGURE 26-5  A, Adult male neutered Rottweiler with a high antibody titer to
tered Boston terrier that was receiving chronic glucocorticoid treatment for a temporal Ehrlichia canis, malnutrition, gastrointestinal parasitism, and sarcoptic mange. B, Oral
lobe glioma. B and C, Histopathology of the lesions revealed hyperkeratosis with increased papillomatosis was present on oral examination and involved the hard and soft palates,
keratohyalin granules and multifocal intracytoplasmic inclusion bodies, as well as second- buccal mucosa, and lips.
ary bacterial infection (not shown). (Courtesy of the University of California, Davis Veteri-
nary Dermatology and Neurology Services.)

FIGURE 26-6  Right thoracic limb digital squamous cell carcinoma in a male neutered
Australian shepherd after amputation. A large, 4 cm × 5 cm ulcerated mass is present.
Immunohistochemistry for papillomavirus was negative, but Canis familiaris papilloma-
virus 2 was detected with PCR and sequencing. (Courtesy of the University of California,
Davis Veterinary Anatomic Pathology Service.)
FIGURE 26-7  Sarcoid of the lip in an 8 year-old female spayed domestic longhair
at necropsy. The cat was euthanized because of oral pain and inability to eat and drink.
(Courtesy of the University of California, Davis Veterinary Anatomic Pathology Service.)
upper layers of the epidermis (see Figure 26-3, C). Endophytic
papillomas are cup-shaped masses of epithelial hyperplasia that
occur below the level of the surrounding normal skin. Koilo-
cytosis and intranuclear inclusions are often prominent. His-
topathology of pigmented plaques are characterized by locally
extensive epidermal hyperplasia, hyperkeratosis, hyperpigmen-
tation, and clumped keratohyalin granules; koilocytes and viral
inclusions are generally not observed.7 Feline sarcoids have der-
mal fibroplastic proliferations with overlying epithelial hyper-
plasia that includes long, thin rete ridges that extend into the
tumor; they may be confused with sarcomas.40 Feline plaques
exhibit moderate to marked epidermal hyperplasia and koilo-
cytosis. Intranuclear inclusions are sometimes present. In situ
SCCs are characterized by epithelial hyperplasia, dysplasia, and
increased numbers of mitotic figures, with an intact basement
membrane. Loss of the basement membrane and invasion of the
dermis indicates invasive SCC.
With the exception of feline sarcoids, immunohistochemis-
try using antibodies that detect conserved papillomavirus anti-
gens (i.e., group-specific papillomavirus antigens) can confirm
the presence of papillomaviruses within lesions. Canine and
feline papillomavirus-specific reagents are not widely available.
CHAPTER 26  Viral Papillomatosis 265
In situ hybridization techniques can also be used. Crystalline
arrays of virus can also be identified within lesions with electron
microscopy.
Treatment and Prognosis
The outcome of papillomavirus infection depends on the virus
strain and host immunity. Oral and cutaneous papillomas in
young dogs generally regress spontaneously 4 to 8 weeks, and
sometimes 3 months after the onset of clinical signs. However,
they may persist in older dogs, or when there is underlying
immune compromise, and occasionally progress to malig-
nancy. If possible, underlying immunosuppression should be
treated (for example, through removal or tapering of immuno-
suppressive drug treatment). Lesions can be removed surgically
if they cause physical obstruction or for cosmetic reasons, but
surgical treatment may be followed by recurrence. Treatment
with azithromycin (10 mg/kg PO q24h for 10 days) appeared
to be effective in a prospective, randomized, double-blinded
placebo-controlled clinical trial that included 12 dogs with
oral papillomatosis and 5 dogs with cutaneous papillomato-
sis.42 The use of topical or intralesional immunomodulators
(such as imiquimod cream) and antiviral drugs (such as cidofo-
vir) has shown promise for treatment of persistent papillomas
in humans, but these treatments have not been evaluated in
dogs and cats.
Pigmented plaques tend to persist, but most are not associated
with malignant transformation. Transformation to a well-differ-
entiated SCC was reported in an affected miniature schnauzer.43
Feline viral plaques have the potential to transform to Bowenoid
SCC in situ and invasive SCCs.32 Feline sarcoids often recur
locally after surgical removal, but metastasis does not occur.
As more information becomes available, certain papillomavirus
types may be more firmly linked to the presence or absence of
malignant transformation, which may aid prognostication.
Immunity and Vaccination
Recovery from oral papillomatosis is associated with papilloma-
virus type-specific resistance to reinfection. Immunity to papil-
lomaviruses comprises (1) the host immune response to the virus
and (2) a cell-mediated immune response to the tumor, which is
ultimately responsible for tumor regression. This explains why
new tumors may not occur, but old tumors persist and continue
to grow in the face of an immune response to the virus, and
immunization with the virus does not induce tumor regression.
Papillomaviruses can evade the host immune response because
infection is limited to the skin, and the immunogenic capsid pro-
tein is not expressed until the virus reaches the terminally differ-
entiated outer layer. A variety of other mechanisms of immune
evasion have been identified in human papillomavirus infec-
tions, such as interference with interferons and inhibition of
chemokines such as Il-8 and Il-18 by papillomavirus proteins.44
Viral persistence is necessary for malignant transformation.
Prevention
No vaccines are available for prevention of papillomatosis in
dogs and cats. Prevention of immunosuppressive conditions,
such as retrovirus infections in cats, and cautious use of potent
immunosuppressive drugs such as cyclosporin can also reduce
the chance of papillomatosis.
266 SECTION 1  Viral Diseases
Public Health Aspects
More than 100 papillomavirus types infect humans. Papilloma-
viruses are relatively species specific, and there is no evidence
that canine papillomaviruses infect humans. However, the DNA
CASE EXAMPLE
Signalment: “Oscar,” an adult male neutered Rottweiler mix
(see Figure 26-5)
History: Oscar was brought to the University of California,
Davis, Veterinary Medical Teaching Hospital for examination
after adoption by a veterinary student as a stray from an
Indian reservation in Arizona. The student was concerned
about the possibility of malnourishment, a painful mouth,
and pruritic skin disease. Since his rescue 5 days previously,
the student had been feeding him four times daily with
maintenance commercial wet and dry dog foods. His
appetite had been ravenous and tapeworm segments had
been observed in his feces. He had been isolated from other
pets in the household.
Physical Examination:
Body Weight: 25.5 kg
General: Bright and alert with normal mentation, nervous.
T = 100.8°F (38.2°C), HR = 90 beats/min, RR = 18 breaths/min,
mucous membranes pink, CRT = 1 s.
Integument: A dull haircoat was present. Patchy generalized
alopecia with severe scaling, crusting, hyperpigmentation,
ulceration, and lichenification was present on the
face, pinnae and pinnal margins, shoulders, trunk, and
extremities.
Eyes, Ears, Nose, and Throat: Moderate mucoid discharge
was present from both eyes. The incisor teeth were
severely worn. Oral examination also revealed pain
on opening the mouth and multiple pink to gray
pedunculated masses that ranged from a few millimeters
to over 1 cm in diameter on the buccal mucosa and
gingiva, lateral aspect of the tongue, and on the hard and
soft palates (see Figure 26-5, B). These were consistent
with oral papillomatosis.
Musculoskeletal: Body condition score 1/9. Normal gait. No
other musculoskeletal abnormalities were noted.
Cardiovascular and Respiratory: No clinically significant
abnormalities were detected.
Gastrointestinal and Genitourinary: Abdominal palpation
was within normal limits. Intact male with soft, symmetrical
testes. Soft feces were noted on rectal examination.
Lymph Nodes: Mandibular and superficial cervical nodes were
enlarged (approximately 3 cm); the popliteal nodes were
sized within normal limits.
Laboratory Findings:
CBC:
HCT 32.3% (40%-55%)
MCV 64.3 fL (65-75 fL)
MCHC 36.2 g/dL (33-36 g/dL)
WBC 10,390 cells/µL (6000-13,000 cells/µL)
Neutrophils 5995 cells/µL (3000-10,500 cells/µL)
Lymphocytes 1891 cells/µL (1000-4,000 cells/µL)
of more than one different human papillomavirus type has been
detected in epithelial lesions in cats.30,31 Further work, includ-
ing whole viral genome sequencing, is required to determine
whether cats can be a source of some papillomavirus infections
for humans and vice versa.
Monocytes 758 cells/µL (150-1200 cells/µL)
Eosinophils 1704 cells/µL (0-1500 cells/µL)
Platelets 322,000 platelets/µL (150,000-400,000 platelets/µL)
Rare macroplatelets observed.
Serum Chemistry Profile:
Sodium 144 mmol/L (145-154 mmol/L)
Potassium 4.8 mmol/L (3.6-5.3 mmol/L)
Chloride 108 mmol/L (108-118 mmol/L)
Bicarbonate 26 mmol/L (16-26 mmol/L)
Phosphorus 5.1 mg/dL (3.0-6.2 mg/dL)
Calcium 10.1 mg/dL (9.7-11.5 mg/dl)
BUN 26 mg/dL ( 5-21 mg/dL)
Creatinine 0.7 mg/dL (0.3-1.2 mg/dL)
Glucose 108 mg/dL (64-123 mg/dL)
Total protein 7.7 g/dL (5.4-7.6 g/dL)
Albumin 2.9 g/dL (3.0-4.4 g/dL)
Globulin 4.8 g/dL (1.8-3.9 g/dL)
ALT 48 U/L (19-67 U/L)
AST 30 U/L (19-42 U/L)
ALP 117 U/L (21-170 U/L)
Gamma GT <3 U/L (0-6 U/L)
Cholesterol 210 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.048; pH 5.0, negative protein (SSA), 1+
bilirubin; no hemoprotein, ketones or glucose; 1-2 WBC/HPF,
rare RBC/HPF, rare bilirubin crystals, moderate sperm.
Cytology of Superficial and Deep Skin Scrapings: Sarcoptes
mites were seen.
Imaging Findings: Lateral and dorsoventral thoracic
radiographs were unremarkable. Abdominal
ultrasonography revealed a small, 1 cm hypoechoic nodule
in the body of the spleen. The remainder of the abdominal
structures was within normal limits.
Microbiologic Testing: Lateral flow ELISA assay for
antibodies to Ehrlichia canis, Anaplasma phagocytophilum,
and Borrelia burgdorferi and Dirofilaria immitis antigen (4Dx
SNAP, IDEXX Laboratories, ME, USA): Positive for antibodies
to E. canis.
Vector-borne disease serology panel (Babesia canis, A. phagocy-
tophilum, E. canis, Rickettsia spp. IFA): Positive for antibodies
to E. canis (1:655,360) and Rickettsia spp. (1:40,960).
Fecal flotation: Giardia spp. cysts (<1 per 40× field), Sarcoptes
mites (1 to 10 per 10× field).
Diagnosis: Canine oral papillomatosis; malnutrition; sarcoptic
mange; suspected chronic canine monocytic ehrlichiosis;
previous exposure to spotted fever group rickettsias;
giardiasis; cestodiasis.
Treatment: Oscar was treated with doxycycline (5 mg/kg
PO q12h for 6 weeks) for ehrlichiosis, topical selamectin
(every 2 weeks for a total of 6 weeks) for sarcoptic
mange, praziquantel (5 mg/kg, once) for cestodiasis,
cephalexin (22 mg/kg q12h for 4 weeks) for secondary
bacterial pyoderma, and fenbendazole (50 mg/kg PO

q24h for 3 days) for giardiasis. The owner was instructed
to bathe him with an oatmeal shampoo twice weekly.
At a recheck appointment 4 weeks later, Oscar weighed
31.5 kg. The skin lesions had improved, and new hair
growth was noted. Pruritus was still present. More oral
papillomas were present, and the owner described
worsened halitosis. Mandibular lymph nodes remained
enlarged. Treatment with cephalexin and doxycycline was
continued for an additional 3 weeks. Three weeks later,
the dog’s bodyweight was 33.4 kg, the oral papillomas
and mandibular lymphadenomegaly had resolved, and
Oscar was vaccinated with canine distemper virus, canine
SUGGESTED READING
Lange CE, Favrot C. Canine papillomaviruses. Vet Clin North Am
Small Anim Pract. 2011;41:1183-1195.
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CHAPTER 26  Viral Papillomatosis 267
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Comments: The gross appearance of the oral lesions in this
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E. canis infection, but the extent to which ehrlichiosis
contributed to papillomatosis in this dog is unknown. The
immunosuppression associated with malnutrition may also
have played a role. Resolution of lesions occurred within
7 weeks after treatment of the immunosuppressive disorders,
but papillomatosis can also resolve spontaneously over the
same time period.
15. Luff JA, Affolter VK, Yeargan B, et al. Detection of six novel papil-
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16. Delius H, Van Ranst MA, Jenson AB, et  al. Canine oral pap-
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17. Lange CE, Tobler K, Ackermann M, et al. Three novel canine pap-
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19. Tobler K, Favrot C, Nespeca G, et al. Detection of the prototype
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20. Tobler K, Lange C, Carlotti DN, et al. Detection of a novel pap-
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21. Yuan H, Ghim S, Newsome J, et  al. An epidermotropic canine
papillomavirus with malignant potential contains an E5 gene and
establishes a unique genus. Virology. 2007;359:28-36.
22. Bregman CL, Hirth RS, Sundberg JP, et al. Cutaneous neoplasms
in dogs associated with canine oral papillomavirus vaccine. Vet
Pathol. 1987;24:477-487.
23. Teifke JP, Lohr CV, Shirasawa H. Detection of canine oral papil-
lomavirus-DNA in canine oral squamous cell carcinomas and p53
overexpressing skin papillomas of the dog using the polymerase
chain reaction and non-radioactive in situ hybridization. Vet
Microbiol. 1998;60:119-130.
24. Zaugg N, Nespeca G, Hauser B, et al. Detection of novel papillo-
maviruses in canine mucosal, cutaneous and in situ squamous cell
carcinomas. Vet Dermatol. 2005;16:290-298.
25. Munday JS, Knight CG. Amplification of feline sarcoid-associated
papillomavirus DNA sequences from bovine skin. Vet Dermatol.
2010.
26. Terai M, Burk RD. Felis domesticus papillomavirus, isolated from
a skin lesion, is related to canine oral papillomavirus and contains
a 1.3 kb non-coding region between the E2 and L2 open reading
frames. J Gen Virol. 2002;83:2303-2307.
27. Munday JS, Willis KA, Kiupel M, et al. Amplification of three dif-
ferent papillomaviral DNA sequences from a cat with viral plaques.
Vet Dermatol. 2008;19:400-404.
28. Nespeca G, Grest P, Rosenkrantz WS, et al. Detection of novel pap-
illomaviruslike sequences in paraffin-embedded specimens of inva-
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2006;67:2036-2041.
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29. Schwittlick U, Bock P, Lapp S, et al. [Feline papillomavirus infec-
tion in a cat with Bowen-like disease and cutaneous squamous cell
carcinoma]. Schweiz Arch Tierheilkd. 2011;153:573-577.
30. Munday JS, Kiupel M, French AF, et al. Detection of papillomavi-
ral sequences in feline Bowenoid in situ carcinoma using consensus
primers. Vet Dermatol. 2007;18:241-245.
31. Anis EA, O’Neill SH, Newkirk KM, et  al. Molecular character-
ization of the L1 gene of papillomaviruses in epithelial lesions
of cats and comparative analysis with corresponding gene
sequences of human and feline papillomaviruses. Am J Vet Res.
2010;71:1457-1461.
32. Munday JS, Peters-Kennedy J. Consistent detection of Felis domes-
ticus papillomavirus 2 DNA sequences within feline viral plaques.
J Vet Diagn Invest. 2010;22:946-949.
33. Munday JS, Howe L, French A, et al. Detection of papillomaviral
DNA sequences in a feline oral squamous cell carcinoma. Res Vet
Sci. 2009;86:359-361.
34. Munday JS, Knight CG, French AF. Evaluation of feline oral
squamous cell carcinomas for p16CDKN2A protein immuno-
reactivity and the presence of papillomaviral DNA. Res Vet Sci.
2011;90:280-283.
35. Doorbar J. The papillomavirus life cycle. J Clin Virol. 2005;32(suppl
1):S7-15.
36. Moody CA, Laimins LA. Human papillomavirus oncoproteins:
pathways to transformation. Nat Rev Cancer. 2010;10:550-560.
37. Munday JS, Aberdein D. Loss of retinoblastoma protein, but not
p53, is associated with the presence of papillomaviral DNA in
feline viral plaques, Bowenoid in situ carcinomas, and squamous
cell carcinomas. Vet Pathol. 2012;49(3):538-545.
38. Stokking LB, Ehrhart EJ, Lichtensteiger CA, et  al. Pigmented
epidermal plaques in three dogs. J Am Anim Hosp Assoc.
2004;40:411-417.
39. Munday JS, O’Connor KI, Smits B. Development of multiple pig-
mented viral plaques and squamous cell carcinomas in a dog infected
by a novel papillomavirus. Vet Dermatol. 2011;22:104-110.
40. Schulman FY, Krafft AE, Janczewski T. Feline cutaneous fibropap-
illomas: clinicopathologic findings and association with papillomavirus
infection. Vet Pathol. 2001;38:291-296.
41. Wilhelm S, Degorce-Rubiales F, Godson D, et  al. Clinical, his-
tological and immunohistochemical study of feline viral plaques and
Bowenoid in situ carcinomas. Vet Dermatol. 2006;17:424-431.
42. Yagci BB, Ural K, Ocal N, et  al. Azithromycin therapy of papil-
lomatosis in dogs: a prospective, randomized, double-blinded,
placebo-controlled clinical trial. Vet Dermatol. 2008;19:194-198.
43. Nagata M, Nanko H, Moriyama A, et al. Pigmented plaques asso-
ciated with papillomavirus infection in dogs: is this epidermodys-
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44. Kanodia S, Fahey LM, Kast WM. Mechanisms used by human
papillomaviruses to escape the host immune response. Curr Cancer
Drug Targets. 2007;7:79-89.
CHAPTER 27
Vector-borne and Other Viral Encephalitides
Jane E. Sykes
Overview of Vector-borne Encephalitides
in Dogs and Cats
First Described: Eastern equine encephalitis, 1831 (horses,
Massachusetts)1; West Nile virus infection, 1937 (human,
Uganda)2; tick-borne encephalitis virus, 1931 (humans,
Austria)3
Causes: RNA viruses that belong to the families Togaviridae
(genus Alphavirus), such as eastern equine encephalitis
virus; Bunyaviridae, such as La Crosse virus; and Flaviviri-
dae, such as West Nile virus and tick-borne encephalitis
virus
Affected Hosts: A variety of animal species including horses,
humans, and dogs
Geographic Distribution: Follows that of the vector host. East-
ern equine encephalitis virus is found in the eastern and
southeastern United States, as well as Central and South
America; West Nile virus is found in Africa, Asia, Middle
East, Europe, and the United States; tick-borne encephali-
tis virus is found in continental Europe and Asia
Mode of Transmission: Primarily mosquitos and for tick-borne
encephalitis virus, Ixodes ticks.
Major Clinical Signs (Dogs): Fever (sometimes >105°F
[40.6°C]), lethargy, neurologic signs, and sometimes sys-
temic signs such as anorexia and diarrhea
Differential Diagnoses: Rabies, distemper, pseudorabies;
bacterial, protozoal, or fungal meningitis; granulomatous
meningoencephalitis; hemic neoplasia with central ner-
vous system involvement
Human Health Significance: Humans are susceptible to
disease caused by many arboviruses. Dogs and cats are
dead-end hosts and do not serve as reservoirs for human
infection. Blood and tissues of affected dogs and cats
should be handled with caution.
Etiology, Epidemiology, and Clinical Features
A large number of viruses that are spread by arthropod vec-
tors (sometimes referred to as arboviruses, for arthropod-borne
viruses) can infect and cause encephalitis in dogs and cats
(Box 27-1 and Table 27-1). These viruses are all enveloped
RNA viruses that are unstable in the environment and readily
inactivated with routine disinfectants. Disease is sporadic and
most infected dogs and cats show no clinical signs of illness.
For many arboviruses, only subclinical infections have been
documented in dogs and cats. Arboviruses are maintained in
vectors and small rodent and/or avian reservoir hosts, and some
of these reservoir hosts can develop illness in association with
infection. Dogs and cats, as well as other domestic animal and
human hosts, are generally considered “dead end” hosts and
do not maintain sufficient viremia to infect arthropods (Figure
27-1). Arboviruses replicate within the arthropod vector and
are transmitted to vertebrate hosts when the vector feeds; this
is followed by viremia and, in some cases, viral spread to the
central nervous system (CNS) and infection of neurons (neuro-
invasion), with subsequent neuronal necrosis and inflammation.
When disease occurs, it has important public health significance,
because the clinical signs can mimic those of rabies. Because
humans have the potential to be infected with these viruses,
serologic evidence of infection of dogs and cats can also have
public health significance.
Alphavirus Infections (Family Togaviridae)
Alphaviruses are mosquito-borne viruses that belong to the fam-
ily Togaviridae. Members of this group that are of importance
to human and animal health include Eastern, Western, and
Venezuelan encephalitis viruses, which primarily cause disease
in humans and horses in the Americas; and Ross River virus,
which causes disease in humans in Australia and the Pacific
islands. Although there is one possible report of encephalitis in
a dog due to Venezuelan equine encephalitis virus,4 only eastern
equine encephalitis virus (EEEV) is a significant cause of disease
in dogs. Other alphaviruses cause subclinical seroconversion
when they infect dogs; in some reports, seropositive cats have
also been identified.5 Dogs have been used as sentinels for the
presence of the virus in epidemiologic serosurveys.6
EEEV is endemic in swampy areas of the southeastern United
States,7 where it is maintained in a variety of avian reservoir
hosts. It causes disease in humans, horses, dogs, and other
domestic animal species. Different viral lineages exist that vary
in pathogenicity for humans and horses. Human cases occur in
the southeastern, eastern, and Great Lakes regions of the United
States, but especially Florida, Georgia, Massachusetts, and New
Jersey (Figure 27-2, A).8 Most affected dogs are from Georgia,
but one affected dog was from New York state.9 Dogs with
signs of encephalitis have always been young (<7 months of
age). Disease usually occurs in late spring or summer. Clinical
signs include fever, lethargy, inappetence, and diarrhea, with
rapid progression (within 1 to 2 days) to recumbency, tachy-
pnea, miosis, nystagmus, tremors, seizures, and death.7
Bunyavirus Infections
Important bunyaviruses for animal and human health are the
mosquito-borne viruses Rift Valley fever (genus Phlebovirus)
and La Crosse (genus Bunyavirus) viruses; the tick-borne virus
269
270 SECTION 1  Viral Diseases

TABLE 27-1
Potential Causes of Vector-borne Viral and Other Viral Encephalitides in Dogs and Cats
Virus Classification Vector Affected Hosts Geographic Distribution
Eastern equine Family Toga- Mosquitos Horses, humans, dogs, Southeastern (especially Georgia and Florida),
encephalitis viridae, genus other domestic farm eastern, and Great Lakes regions of the
virus Alphavirus animal species United States
Venezuelan Family Toga- Mosquitos Horses, humans, ­possibly Southern United States (Florida), Central and
equine en- viridae, genus dogs South America (especially Colombia, Ven-
cephalitis Alphavirus ezuela, Trinidad, Peru, Ecuador, Mexico)
virus
La Crosse virus Family Bunyaviri- Mosquitos Humans, dogs Eastern and Midwestern United States
dae
West Nile virus Family Flaviviridae Mosquitos Horses, humans, birds, North, Central, and South America; Africa, the
dogs Middle East, Europe, Asia, Australia
Tick-borne Family Flaviviridae Ixodes ticks Humans, horses, dogs; Europe, Asia
encephalitis domestic animals are
virus generally infected
­subclinically
Borna disease Family Bornaviri- None known Horses, cattle, cats, dogs Primarily Sweden, Austria, Germany, and
virus dae Japan

La Crosse virus was named after La Crosse in Wisconsin, where

BOX 27-1
General Features of Arbovirus Infections as They Relate
to Dogs and Cats
Vector-borne enveloped RNA viruses
Most often mosquito-borne
Maintained among small wild vertebrate reservoir hosts,
especially birds and/or rodents
Geographic distribution follows that of vector host
Seasonal distribution that reflects activity of the vector
host
May cause disease in humans and other domestic animals,
especially horses
Infection of dogs and cats is most often subclinical
Dogs and cats are “dead end” hosts
Infection more prevalent in dogs than in cats
Sporadic reports of disease, especially in young animals
(<6 months)
Neurologic signs accompanied by fever and sometimes
other systemic signs (e.g., diarrhea)
Histopathology shows neuronal necrosis, gliosis, and
perivascular cuffing within the brain, which is predomi-
nantly nonsuppurative.
it was first identified as a human pathogen, and is found mostly
in the eastern and midwestern United States (see Figure 27-2, B).
The virus is maintained in rodents, especially Eastern gray
squirrels and chipmunks. It is a major cause of human pediatric
encephalitis in North America, although mortality is low.
Affected dogs have included an adult dog from Florida and
three puppies from southern Georgia.12,13 Mild fever, lethargy,
ataxia, vestibular signs, and ultimately seizures occurred in the
adult dog, and sudden death was reported in the puppies.
Flavivirus Infections
Vector-borne flaviviruses (flavi = “yellow,” Latin) of impor-
tance to human and animal health are the mosquito-borne
viruses Japanese encephalitis virus, yellow fever virus, Murray
Valley encephalitis virus, St. Louis encephalitis virus, Wessels-
bron virus, Dengue viruses, and West Nile virus (WNV), as well
as the tick-borne viruses tick-borne encephalitis (TBE) virus,
louping ill virus, and Powassan virus. Antibodies to some of
these viruses have been detected in dogs and to a lesser extent,
cats in serosurveys,14-18 but serologic cross-reactivity among fla-
viviruses can occur. Although uncommon to rare, WNV in the
United States and TBE virus in Europe have been reported to
cause disease in dogs on more than one occasion. One dog from
Africa with fatal encephalitis was initially thought to be infected
with Wesselsbron disease virus,19 but the isolate was later con-
firmed to be WNV.20

Crimean-Congo hemorrhagic fever virus (genus Nairovirus);
and members of the genus Hantavirus, which are not transmit-
ted by vectors and cause hemorrhagic febrile and respiratory ill-
ness in humans. Serologic evidence of infection with some these
viruses has been documented in dogs and cats.10,11 La Crosse
virus is the only bunyavirus reported to cause disease in dogs
under natural circumstances, but this appears to be rare.12,13
West Nile Virus Infection
West Nile virus was first isolated in 1937 from a woman in
the West Nile region of Uganda.21 In the 1950s, the virus
was recognized in Israel as a cause of human neurologic dis-
ease in the elderly,22 and subsequently was identified in other
parts of Europe and South Africa. In 1999, it appeared in New
York state, possibly by transport of infected humans, birds, or
 CHAPTER 27  Vector-borne and Other Viral Encephalitides 271

FIGURE 27-1  West Nile virus transmission cycle and factors that influence arbovirus epidemiology.

A B

Western European Tick-borne Encephalitis

Eastern European Tick-borne Encephalitis
D Overlapping Distribution
FIGURE 27-2  Geographic distribution of some arboviruses that can cause disease in dogs and cats. A, Regions with eastern equine encephalitis virus transmission. B, Regions with
La Crosse virus transmission. C, Regions with West Nile virus transmission. D, Regions with tick-borne encephalitis virus transmission.

mosquitos.23 WNV then spread explosively westward across
the United States, and by 2003, disease was detected across
the United States to California (see Figure 27-2, C). WNV also
spread into Canada, Mexico, the Caribbean, and Central and
South America.23,24 Several different variants of the virus exist,
which differ in virulence. Strains circulating within the Western
hemisphere, Europe, the Middle East, India, Africa, and Austra-
lia belong to WNV lineage 1; lineage 2 strains circulate in South
Africa and Madagascar. Other lineages have also been reported.
Transmission of WNV by mosquitos increases in the warmer
months, with peak activity between July and October. Birds
(especially passerines such as crows, jays, and brown sparrows;
272 SECTION 1  Viral Diseases
transstadial
transmission
bank voles
field mice
larva
cofeeding
transstadial
transmission
eggs
transovarial
Ixodes ricinus
transmission
adult tick
FIGURE 27-3  Transmission cycle for tick-borne encephalitis virus.
shorebirds; owls; and hawks) are important reservoir hosts (see
Figure 27-1). Some avian species, such as brown sparrows,
maintain high viremias in the absence of clinical signs. Other
modes of transmission occur, such as ingestion through preda-
tion of infected birds or rodents, blood transfusion, needle-stick
injuries, and transplacental transmission. Disease primar-
ily occurs in birds, humans, and horses. When signs occur in
humans they are often mild and self-limiting and include fever,
headache, myalgia, fatigue, gastrointestinal symptoms, and a
transient rash.25 Rarely, hepatitis, pancreatitis, myocarditis,
rhabdomyolysis, orchitis, uveitis, and chorioretinitis can occur.
Severe, neuroinvasive disease occurs in fewer than 1% of cases,
is most common in the elderly and results in high fever, cervical
rigidity, muscle weakness, seizures, and flaccid paralysis. Neu-
rologic signs result from viral invasion of neurons in the brain-
stem, deep nuclei, and anterior horns within the spinal cord.
Clinical signs in horses result from polioencephalomyelitis and
include fever and neurologic signs such as ataxia, weakness, and
muscle tremors.26 Approximately two thirds of horses recover.
Infected wild birds can exhibit neurologic disease but are usu-
ally found dead. Myocarditis and meningoencephalitis are com-
mon necropsy findings in these birds.27
In dogs, there is widespread serologic evidence of infection,
but disease is rare. Cats can become infected and seroconvert,
but illness has not been described. Five clinically affected dogs
have been described. All, apart from the dog from Africa,
resided in various parts of the United States (Missouri, Mis-
sissippi, California, and Illinois).28-31 All of the dogs had signs
of fever and progressive neurologic disease. Neurologic signs
included generalized tremors, ataxia, a head tilt, bobbing move-
ments, altered mentation, cervical pain, abnormal placing reac-
tions, and tetraparesis. Diarrhea, serous oculonasal discharge,
conjunctivitis, abdominal pain, cardiac arrhythmias due to
myocarditis, tachypnea, tachycardia, and a stiff gait associated
with mild neutrophilic polyarthritis have been reported. For all
dogs, diagnosis was made at necropsy.
nymph
Tick-Borne Encephalitis Viruses
TBE virus is a notifiable disease that causes potentially fatal
neurologic disease in humans in Europe and Asia, where it is
the most important tick-borne viral disease of humans.32 The
virus is transmitted transstadially and to a lesser extent trans-
ovarially within infected ticks, and ticks can transmit the dis-
ease to each other while co-feeding, independent of systemic
viremia in vertebrate hosts.33 Consumption of raw milk from
infected ruminants is a lesser mode of transmission to humans
(Figure 27-3).
Three subtypes of TBE virus exist, which vary in virulence
and geographic distribution. These are designated the Euro-
pean, Siberian, and far-eastern subtypes.34 The far-eastern sub-
type occurs in far eastern Russia, Japan, Korea, and China and
is the most virulent subtype, with mortality rates in humans
that may exceed 20%. In much of Europe, Ixodes ricinus
ticks spread the European subtype (Western European TBE)
whereas Ixodes persulcatus ticks transmit the other viral sub-
types in northeastern Europe and Asia (Eastern European TBE)
(Figure 27-2, D).35 Both Siberian and European subtypes may be
found in eastern Europe. The disease was originally detected in
1937 in Austria, but its geographic distribution has expanded rap-
idly so that the European subtype now occurs throughout Europe
as far west as France and possibly Belgium.17 In Europe, some
of the highest incidence rates have occurred in central European
countries, especially the Czech Republic.36 Disease seasonality cor-
relates with the activity of the tick in various parts of Europe. TBE
has not been reported in the United Kingdom, but a related tick-
borne flavivirus that mainly causes disease in sheep, Louping ill
virus, is present. This virus rarely causes encephalitis in dogs.37,38
In humans, signs of TBE occur in fewer than 0.5% of
infected individuals 4 to 28 days after a tick bite and are
biphasic. In the first, viremic phase, fever, malaise, headache,
myalgia, and vomiting occur, which resolve within 1 week. In
some individuals, after an asymptomatic period that lasts 2 to
10 days, clinical signs return and are accompanied by signs of
 CHAPTER 27  Vector-borne and Other Viral Encephalitides
meningitis, meningoencephalitis, meningoencephalomyelitis,
or meningoencephaloradiculitis. About 1% of cases are fatal,
most often in the elderly,39 but neurologic signs can persist
for months or longer than a year. Co-infections with Borrelia
burgdorferi can occur. Vaccines are currently recommended
for prevention of TBE in humans, and national vaccination
programs in Austria have greatly reduced the incidence of
disease.40
Subclinical infection of dogs with TBE virus is widespread,
and so dogs have been important sentinels for the presence of
the virus in new regions of Europe. Rarely, illness that resem-
bles that in humans can occur in dogs. Affected dogs have been
described from Sweden, Switzerland, Austria, Germany, and
Italy.32,41 Several reports have described disease in Rottweilers,
but it is not clear whether this represents a true breed predis-
position. Signs in dogs include high fever, lethargy, anorexia,
and neurologic signs such as decreased mentation, behavioral
changes such as aggression, delayed placing reactions, ataxia,
flaccid paralysis, reduced segmental reflexes, neck pain, vestibu-
lar signs, tremors, tetraparesis, miosis, anisocoria, and general-
ized seizures.32,35 These signs progress rapidly over several days
to a week, usually culminating in death or euthanasia. Recovery
has been described rarely.
Bornaviridae
Borna disease virus (BDV) is an enveloped RNA virus that causes
meningoencephalitis primarily in horses in Europe, but also in
sheep, cattle, and a variety of other domestic and wild animal
species. On rare occasions, cats and dogs are affected.42,43 BDV
has been suggested as a possible but unconfirmed cause of neu-
ropsychiatric illness in humans.44 The epidemiology of BDV
remains mysterious. There is some evidence that small mammals
(rodents) act as reservoir hosts. In one study, cats at risk were
intact male cats, cats that hunted, and cats that resided in a rural
habitat.45 BDV can be transmitted horizontally among labora-
tory rodents via urine.46 Because of seasonal disease occur-
rence in the spring and summer, an arthropod vector has been
suspected, but like rabies virus, BDV is thought to enter the
CNS principally via axonal transport from mucosal surfaces,
which suggests that direct transmission may be more important.
Entrance via the olfactory system has been suggested based on
lesions found in horses.44
Feline infection was first recognized in Sweden in the early
1970s. The virus has also been detected in cats with neuro-
logic disease from Austria, Switzerland, the United Kingdom,
and Japan.47-49 Some studies have shown a higher prevalence
of antibodies to BDV in cats with neurologic signs than in cats
without neurologic signs. Affected dogs have been reported
from Japan and Austria.42,43 Serologic evidence of infection has
been detected in a low percentage (3.7%) of cats that lacked
neurologic disease from the western United States.50
The clinical signs of BDV infection can be explained by
the predilection of the virus for the limbic system, brainstem,
and basal ganglia. In cats, the disease has been referred to as
“staggering disease” and is manifested by behavioral changes
and increased vocalization, anorexia, lethargy, fever, ataxia,
posterior paresis, inability to retract the claws, hypersaliva-
tion, and seizures. Clinical signs generally progress over 1 to
6 weeks, which is followed by death or euthanasia or perma-
nent residual neurologic dysfunction. Affected dogs have had
anorexia, lethargy, tremors, salivation, mydriasis, circling, and
seizures.42,43
273
Diagnosis
Laboratory Abnormalities
Laboratory abnormalities in dogs with vector-borne viral
encephalitis are nonspecific. No or minimal abnormalities may
be detected in some dogs. Mild anemia, leukopenia, lympho-
penia, or leukocytosis due to a neutrophilia (sometimes with
a left shift) may occur. The CSF may also be unremarkable, or
there may be increased protein concentration and mononuclear
pleocytosis.
Microbiologic Tests
Serologic Diagnosis
Serologic assays such as ELISAs are available for detection of
antibodies to arboviruses through specialized veterinary diag-
nostic or research laboratories, and have primarily been used
for epidemiologic serosurveys of dogs and cats. Serologic assays
have, in general, not been very useful for diagnosis of acute
viral encephalitis in dogs and cats. First, because disease is rare,
arbovirus infections are often not suspected before death or
euthanasia, and euthanasia is often performed early because of
suspicion for rabies. Second, incubation periods are often short
and disease in dogs progresses rapidly with a high mortality
rate, so there is insufficient time for an antibody response to
develop. Serologic assays have mainly been used to detect IgG
and IgM in dogs with TBE from Europe.32
Virus Isolation
Arboviruses can be isolated in a variety of cell lines, and virus
isolation has been used to detect WNV, TBE virus, and
La Crosse virus in naturally infected dogs with encephalitis
and BDV in cats.12,28,47,51 Fresh or fresh-frozen brain or spi-
nal cord homogenates are suitable specimens. Because viremia
occurs, blood, CSF, lung, spleen, and liver might also be useful
specimens for isolation.28,32 Immunofluorescent antibody stain-
ing and reverse transcriptase–PCR (RT-PCR) assays are used to
confirm the presence of the virus in cell cultures.7,28,47 Because
these viruses can infect humans, specialized biosafety level 3
facilities are generally required. This limits the availability and
practicality of virus isolation for routine diagnostic purposes.
Molecular Diagnosis Using the Polymerase Chain Reaction
Conventional and real-time RT-PCR assays have been described
for many different arboviral pathogens and BDV. Specialized
veterinary diagnostic and research laboratories generally per-
form these assays. In dogs, RT-PCR assays have been used to
detect EEEV, WNV, and BDV in clinical specimens.7,28,42,43
Because BDV can be detected using PCR assays in tissues in the
absence of clinical signs, other methods that localize virus to
sites of inflammation, such as immunohistochemistry or in situ
hybridization, are necessary for diagnosis of BDV encephalitis.
Pathologic Findings
In general, histopathology of the CNS in dogs with vector-borne
viral infections, and cats and dogs with Borna disease, reveals
nonsuppurative meningoencephalitis or meningoencephalo-
myelitis, with multifocal mild to moderate gliosis, neuronal
necrosis, and perivascular cuffing with lymphocytes, histio-
cytes, and plasma cells within Virchow-Robin spaces.12,32,43
Neutrophils predominate within the meninges of dogs infected
with EEEV.7 Within brain tissue, inflammatory infiltrates in
dogs infected with EEEV have been most prominent in the gray
274 SECTION 1  Viral Diseases
A A
B
FIGURE 27-4  Spinal cord segment of a dog with WNV encephalitis that shows gliosis
and multiple degenerate neurons. A, H&E stain. B, Immunohistochemical stain showing
WNV immunoreactivity (brown). (From Cannon AB, Luff J, Brault C, et al. Acute encepha-
litis, polyarthritis and myocarditis associated with West Nile virus infection in a dog. J Vet
Internal Med 2006;20:1219-1223.)
matter of the cerebral cortices and midbrains. Gray matter has
also been preferentially infected in dogs with WNV encepha-
litis.28 Pathologic changes in dogs with TBE are most severe
in the brainstem and the cerebellum; meningitis and spinal
cord involvement have also been described.32 BDV preferen-
tially infects the limbic system and brainstem, although the
cerebral cortex may also be involved, and one dog had diffuse
disease.39,40,44 Immunohistochemistry has been used to detect
antigen of WNV, La Crosse virus, and TBE virus in CNS and
other tissues of dogs, and to detect BDV antigen in the CNS
of cats and dogs at necropsy (Figure 27-4).13,28,41-43,47 In situ
hybridization has been used to detect BDV RNA in the CNS of
dogs and cats.43,48
Extraneural lesions have mainly been reported in dogs
infected with WNV. Lymphocytic and necrotizing myocardi-
tis was present in several dogs (Figure 27-5). Other findings in
dogs with WNV infection have included evidence of hepatic and
renal tubular epithelial cell necrosis, neutrophilic pancreatitis,
and mild plasmacytic synovitis.28-31
Treatment and Prognosis
Treatment of dogs with vector-borne viral encephalitis is sup-
portive only. The prognosis appears to be poor, but because
B
FIGURE 27-5  Severe lymphocytic and neutrophilic myocarditis and vasculitis due
to WNV infection with focally extensive hemorrhage and myonecrosis. A, H&E stain. B,
Immunohistochemical stain showing WNV immunoreactivity within neutrophils and his-
tiocytes (brown). (From Cannon AB, Luff J, Brault C, et al. Acute encephalitis, polyarthri-
tis and myocarditis associated with West Nile virus infection in a dog. J Vet Internal Med
2006;20:1219-1223.)
only a handful of infected dogs have been reported, the true
prognosis is poorly understood. In virtually all dogs with neu-
rologic signs, diagnosis has been made at necropsy. Some dogs
with TBE recover with supportive care.32 Glucocorticoids have
been used to treat some dogs and cats with viral encephalitides,
but their use is controversial because of immunosuppression,
and controlled studies are not available.
Immunity and Vaccination
Exposure to vector-borne viruses appears to confer immunity to
future infections with the same virus. Vaccines are not available
for dogs because disease is uncommon to rare. However, vac-
cines are available and used widely in endemic areas to prevent
flavivirus infections in humans (such as TBE, yellow fever, and
Japanese encephalitis) and WNV encephalitis in horses.
Prevention
The risk of arboviral infections in dogs and cats can be reduced
through control of mosquito and tick vectors. Keeping pets
indoors, especially at dawn and dusk when mosquitos are
most active, can also reduce exposure, provided mosquitos
are excluded through the use of proper screens and windows.
 CHAPTER 27  Vector-borne and Other Viral Encephalitides
For viruses such as WNV and BDV, prevention of predation
on dead birds or rodents may also reduce the risk of infec-
tion. Drainage of standing water and removal of containers
(tin cans, old tires, flower pots, clogged gutters, bunched tar-
paulins) that act as breeding sites are important for mosquito
control. Water in birdbaths should be changed at least weekly.
Weeds and brush should be reduced. Human mosquito repel-
lents that contain DEET are not recommended for pets, because
they can be toxic.52 Some products labeled for tick prevention
in dogs and cats may help to repel mosquitos but are not likely
to completely protect against mosquito bites. More information
on tick prevention can be found in Chapter 28. Only products
CASE EXAMPLE
Signalment: “Paloma,” a 13-year-old female spayed golden
retriever–Irish setter mix from Vacaville in northern
California.
History: Paloma was evaluated at the University of California,
Davis, Veterinary Medical Teaching Hospital in September
for a 3-day history of inappetence and lethargy. Two days
before, physical examination by the referring veterinarian
had revealed tachycardia (96 beats/min) and pyrexia (103.1°F
or 39.5°C). Results of routine CBC, biochemistry tests, and a
urinalysis did not reveal clinically significant abnormalities.
The dog was treated with amoxicillin (20 mg/kg PO q12h)
and enrofloxacin (10 mg/kg PO q12h), without improvement.
Physical Examination:
Body Weight: 28.6 kg
General: Obtunded, 5% dehydrated. T = 106°F (41.1°C),
HR = 140 beats/min, RR = 92 breaths/min, mucous
membranes pink, CRT = 1 s.
Integument: No clinically significant abnormalities.
Eyes, Ears, Nose, and Throat: Bilateral enophthalmos,
mild serous ocular discharge, conjunctivitis, and nuclear
sclerosis were present. Moderate to severe dental disease
was present. Examination of the ocular fundus showed no
abnormalities.
Musculoskeletal: Body condition score 4/9. Ambulatory on all
four limbs, but generalized weakness was noted. Moderate
atrophy of the temporalis, quadriceps, and gastrocnemius
muscles was present.
Cardiovascular, Respiratory, Gastrointestinal, Urogenital
and Lymphoid Systems: Tachypnea and tachycardia were
identified. There was mild cranial abdominal discomfort on
palpation. All peripheral lymph nodes were normal sized.
Neurologic Examination: Decreased placing reactions were
noted in all four limbs, and there was pain on manipulation
of the neck.
Laboratory Findings:
CBC:
HCT 37.5% (40%-55%)
MCV 67.3 fL (65-75 fL)
MCHC 34.4 g/dL (33-36 g/dL)
WBC 6050 cells/µL (6000-13,000 cells/µL)
Neutrophils 5627 cells/µL (3000-10,500 cells/µL)
Lymphocytes 363 cells/µL (1000-4000 cells/µL)
Monocytes 61 cells/µL (150-1200 cells/µL)
Adequate platelet numbers (clumped on smear).
275
specifically labeled for cats should be used on cats or on dogs
that are in contact with cats, because of the sensitivity of cats to
permethrin toxicosis.53
Public Health Aspects
Many arboviruses that cause disease in humans also infect dogs
and cats. Dogs and cats are generally not considered reservoirs
for transmission of virus to humans because they do not develop
high-level or sustained viremia. As noted previously, dogs have
been used as sentinels for the presence of these viruses in epide-
miologic studies.
Serum Chemistry Profile:
Sodium 144 mmol/L (145-154 mmol/L)
Potassium 4.0 mmol/L (3.6-5.3 mmol/L)
Chloride 110 mmol/L (108-118 mmol/L)
Bicarbonate 16 mmol/L (16-26 mmol/L)
Phosphorus 3.1 mg/dL (3.0-6.2 mg/dL)
Calcium 9.1 mg/dL (9.7-11.5 mg/dl)
BUN 8 mg/dL (5-21 mg/dL)
Creatinine 0.6 mg/dL (0.3-1.2 mg/dL)
Glucose 86 mg/dL (64-123 mg/dL)
Total protein 5.7 g/dL (5.4-7.6 g/dL)
Albumin 2.3 g/dL (3.0-4.4 g/dL)
Globulin 3.4 g/dL (1.8-3.9 g/dL)
ALT 25 U/L (19-67 U/L)
AST 34 U/L (19-42 U/L)
ALP 53 U/L (21-170 U/L)
Gamma GT 3 U/L (0-6 U/L)
Cholesterol 236 mg/dL (135-361 mg/dL)
Total bilirubin 0.3 mg/dL (0-0.2 mg/dL).
Imaging Findings:
Thoracic, Abdominal, Spinal, and Joint Radiographs: No
significant abnormalities were noted.
Abdominal Ultrasound: Mild mesenteric and moderate
sublumbar lymphadenomegaly was identified. The dog
exhibited pain on ultrasound of the right cranial abdomen, but
no abnormalities in this region were identified. Impressions:
mesenteric and sublumbar lymphadenomegaly.
Microbiologic Testing: Aerobic and anaerobic blood
cultures (five sequentially collected specimens): negative
Serology for Ehrlichia canis, Anaplasma phagocytophilum, and
Borrelia burgdorferi antibodies (lateral flow ELISA): negative
Treatment: Paloma was treated with lactated Ringer’s solution
(3 mL/kg/h IV) with potassium chloride supplementation
(20 mEq/L), amoxicillin (22 mg/kg PO q8h), enrofloxacin
(10 mg/kg PO q24h), and doxycycline (5 mg/kg PO q12h).
Physical examination on day 2 revealed persistent pyrexia
(105.6°F), tachycardia (108 beats/min), and a stiff gait, but
there was no evidence of joint pain or swelling on palpation.
Cytologic analysis of synovial fluid collected from both
carpal and tibiotarsal joints using arthrocentesis revealed
mild suppurative and mononuclear inflammation.
On day 3, the dog became tetraparetic and was unable to stand
for longer than a few seconds. Pyrexia (104.5°F) persisted,
and treatment with dexamethasone sodium phosphate (0.2
mg/kg IV once) and hydromorphone (0.05 mg/kg IV once)
was initiated. Neurologic examination disclosed mental
dullness, decreased placing reactions in all four limbs, and
276 SECTION 1  Viral Diseases
normal segmental reflexes. Further evaluation for neuro-
logic disease, such as imaging of the brain, CSF analysis, or
both, was offered, but declined by the owner because of
financial limitations. On day 4, the dog was normothermic,
and sternally recumbent. Prednisone (1 mg/kg PO q12h)
and metronidazole (10 mg/kg PO q8h) were added to the
treatment regime. Despite this treatment, by day 5, Paloma
was laterally recumbent and obtunded. The owner elected
euthanasia.
Necropsy Findings: Histopathologic findings at necropsy
consisted of severe acute multifocal necrotizing perivascular
and random neutrophilic pancreatitis; severe lymphocytic
and neutrophilic myocarditis and vasculitis with myonecrosis;
and mild, multifocal gliosis and perivascular to random
lymphoplasmacytic and neutrophilic polioencephalomyelitis.
(From Cannon AB, Luff JA, Brault AC, et al. Acute encephalitis, polyarthritis, and myocarditis associated with West Nile virus infection in a dog. J
Vet Intern Med 2006;20:1219-1223.)
SUGGESTED READINGS
Cannon AB, Luff JA, Brault AC, et al. Acute encephalitis, polyarthritis,
and myocarditis associated with West Nile virus infection in a dog.
J Vet Intern Med. 2006;20:1219-1223.
Farrar MD, Miller DL, Baldwin CA, et al. Eastern equine encephalitis in
dogs. J Vet Diagn Invest. 2005;17:614-617.
Murray KO, Mertens E, Despres P. West Nile virus and its emergence in
the United States of America. Vet Res. 2010;41:67.
Pfeffer M, Dobler G. Tick-borne encephalitis virus in dogs—is this an
issue? Parasit Vectors. 2011;4:59.
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The cervical spinal cord was the most severely affected. Mild
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and anaerobic cultures of the liver, spleen, spleen, lung, and
synovial fluid were negative. West Nile virus antigen was
detected with immunohistochemical stains within foci of
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from fresh frozen lung tissue, and WNV RNA was detected
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heart, and kidney tissue.25
Diagnosis: Encephalitis, myocarditis, and polyarthritis
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 SECTION 2
Bacterial Diseases
CHAPTER 28
Ehrlichiosis
Jane E. Sykes
Overview of Ehrlichiosis
First Described: Ehrlichia canis was first described in 1935
(Algeria).1 Ehrlichia ewingii was described in 1992 (United
States).2 Ehrlichia chaffeensis was described in 1991
(United States).3
Cause: E. canis (canine monocytic ehrlichiosis), E. ­ewingii
(canine granulocytic ehrlichiosis), and E. chaffeensis
(human monocytic ehrlichiosis); an E. muris-like organism
also may infect dogs in the United States.4
Affected Hosts: E. canis causes disease in dogs. E. canis or a
closely related organism may cause disease in cats and
humans. E. ewingii causes disease in dogs, humans, and
goats. E. chaffeensis causes disease in humans, possibly
dogs and goats. The E. muris-like agent causes disease in
humans and possibly dogs.
Geographic Distribution: E. canis is present worldwide, but
especially in tropical and subtropical regions. E. ewingii
is primarily found in the south-central and southeastern
United States. Most reports of E. chaffeensis infection
are from the southern and south-central United States.
The E. muris-like agent has been found in the upper
Midwest.
Mode of Transmission: Rhipicephalus sanguineus ticks (E. canis),
Amblyomma americanum ticks (E. ewingii and E. chaffeensis).
Major Clinical Signs: The major clinical signs of E. canis infec-
tion are fever, lethargy, inappetence, weight loss, mucosal
hemorrhages, uveitis, pallor, edema, and sometimes neu-
rologic signs. E. ewingii primarily causes fever, lethargy,
inappetence, and signs of polyarthritis.
Differential Diagnoses: Other tick-borne diseases (such as
granulocytic anaplasmosis, Lyme borreliosis and babe-
siosis), ­bartonellosis, leptospirosis, lymphoma, multiple
myeloma, systemic primary immune-mediated disease
278
Human Health Significance: All four ehrlichial species can
infect and cause disease in humans. The most important
human pathogen is E. chaffeensis, but the E. muris-like
agent may also be important.
T
he ehrlichioses are a group of tick-transmitted diseases
caused by intracellular, gram-negative bacteria that include
Ehrlichia canis, Ehrlichia ewingii, and Ehrlichia chaffeen-
sis. An organism related to Ehrlichia ruminantium, the cause of
heartwater disease in cattle, has also been detected in ill dogs from
South Africa,5 and an organism that resembles E. muris has been
detected in an ill dog and humans from the upper Midwest of the
United States.4 These organisms form morulae (Latin for “mul-
berry”), a cluster of bacteria, within phagosomes of circulating
leukocytes. Ehrlichia canis infects monocytes and causes canine
monocytic ehrlichiosis (CME), one of the most important infec-
tious diseases of domestic dogs that are exposed to ticks world-
wide. Ehrlichia ewingii is an unculturable bacterium that infects
granulocytes and causes canine granulocytic ehrlichiosis. Ehrlichia
chaffeensis causes human monocytic ehrlichiosis; dogs are a pro-
posed reservoir for this organism. The geographic distribution of
each pathogen is generally restricted to that of their vectors and
mammalian reservoir hosts.
Organisms from the genus Ehrlichia are grouped within
the family Anaplasmataceae. Also within this family are the
bacteria Anaplasma platys and Anaplasma phagocytophilum,
which cause canine thrombocytic and granulocytic anaplasmo-
sis, respectively (see Chapter 29); and organisms belonging to
the genera Neorickettsia (see Chapter 31). Rickettsia rickettsii,
the cause of Rocky Mountain spotted fever (RMSF), and other
spotted fever group rickettsiae belong to a separate family,
the Rickettsiaceae (Chapter 30). The families Rickettsiaceae
and Anaplasmataceae are phylogenetically related through
the order Rickettsiales (Table 28-1). The recent availability
of complete genome sequences for these organisms has helped
 CHAPTER 28  Ehrlichiosis 279

TABLE 28-1
Members of the Order Rickettsiales of Clinical Importance in Dogs and Cats

Family Anaplasmataceae Rickettsiaceae

Genus Ehrlichia Anaplasma Neorickettsia Rickettsia
Species E. canis A. phagocytophilum N. helminthoeca R. rickettsii
E. chaffeensis A. platys N. risticii
E. ewingii

United States of America United States of America

Mexico Mexico
Brown Dog Tick Lone Star Tick
(Rhipicephalus sanguineus) (Amblyomma americanum)
Belize Belize
Guatemala Honduras Guatemala Honduras
El Salvador Nicaragua El Salvador Nicaragua
Costa Rica Costa Rica
A Panama B Panama
FIGURE 28-1  A, Distribution of Rhipicephalus sanguineus, which transmits Ehrlichia canis, in the United States, Mexico and Central America. B, Distribution of Amblyomma america-
num, which transmits Ehrlichia ewingii and Ehrlichia chaffeensis. The distribution of E. ewingii infections in dogs more closely matches that of A. americanum than the distribution of E. canis
matches that of R. sanguineus (highest prevalence in the southern states). This is because of chronic E. canis infections that occur in dogs with travel histories to southern states, where the
climate is warmer and R. sanguineus is more prevalent.

to elucidate mechanisms of pathogenesis and host-pathogen Eggs

No transovarial
interactions.5-7 transmission
The severity of clinical signs in animals with ehrlichial infec-
tions depends on factors such as the size of the inoculum, host
immunity, and organism species and strain. Because of shared
arthropod vectors and/or concurrent exposure to multiple vec-
tor ticks, co-infections with more than one rickettsial pathogen, Adult tick
as well as other arthropod-borne pathogens such as Babesia
spp., and Bartonella spp., occur commonly in dogs and may
Larva
complicate the clinical picture.

Ehrlichia canis Infection

Etiology and Epidemiology
E. canis is transmitted primarily by the brown dog tick
(­Rhipicephalus sanguineus), one of the most widely distrib-
uted ticks worldwide. Infection has been reported in dogs from
Asia, Africa, Europe, and the Americas. Australia appears to be
free of E. canis infection although occasionally seroreactivity to
E. canis has been identified in dogs. The DNA of E. canis has been
detected in other tick species, which include other ­Rhipicephalus
species,8,9 Ixodes ricinus,10 Haemaphysalis spp. ticks,11 and Der-
macentor spp. ticks11; experimental transmission has been accom-
plished with Dermacentor variabilis ticks.12 Different strains of
E. canis exist that may vary in virulence. Although R. sanguineus
is found throughout the United States, it prefers warm climates,
and so disease is diagnosed most frequently in dogs living in the
southeastern and southwestern states (Figure 28-1). In Europe, R.
sanguineus is primarily found in Mediterranean regions, but its
Nymph
FIGURE 28-2  Life cycle of Ehrlichia canis. The organism is transmitted only transsta-
dially (from larva to nymph to adult) within the tick. Jackals, foxes, and possibly coyotes
also act as reservoir hosts. A morula is shown within the cytoplasm of a monocyte as seen
on a blood smear.
distribution appears to be moving northward, and CME has been
reported in dogs that lack travel history as far north as the Nether-
lands.13 E. canis is the most common pathogen detected in ticks in
Israel.8 Because of chronic, subclinical infection, dogs can be trans-
ported to non-endemic regions and subsequently develop disease
years later. Tick larvae or nymphs acquire infection when they
feed on infected dogs. Jackals, foxes, and possibly coyotes may
also act as reservoir hosts. E. canis is transmitted transstadially
280 SECTION 2  Bacterial Diseases
(i.e., from larva to nymph to adult) within the tick (Figure 28-2).14
No clear age or sex predilection for CME exists, but German
shepherds are reportedly more susceptible, and prognosis may be
poorer in this breed. Cross-bred dogs may be less likely to develop
disease.15 Although natural infection of cats with E. canis (or one
or more closely related organisms) has been described in North
and South America,16-18 clinical ehrlichiosis is rarely reported.
Clinical Features
Signs and Their Pathogenesis
The course of CME has been divided into acute, subclinical,
and chronic phases, although in naturally infected dogs, these
phases may not be readily distinguishable. Clinical signs of
acute disease occur 8 to 20 days after infection. The organism
multiplies by binary fission within vacuoles of mononuclear
phagocytes; rupture of infected host cells leads to infection of
new cells. Immune-mediated mechanisms are important in the
­pathogenesis of disease, and the presence of the spleen appears
to ­contribute to disease severity.19 The clinical manifestations
vary considerably among dogs, which may reflect factors such as
E. canis strain variation, host immune response, stage of dis-
ease, and concurrent infections. Lethargy, inappetence, fever,
and weight loss are most common. Replication of the organ-
ism in reticuloendothelial tissues is associated with general-
ized lymphadenopathy and splenomegaly. Ocular and nasal
discharges, peripheral edema, and, less commonly, mucosal
and cutaneous petechial and ecchymotic hemorrhages can also
occur. Bleeding tendencies result from thrombocytopenia and
platelet dysfunction,20 which may reflect immune-mediated
platelet damage.21 Neurologic signs may result from menin-
geal inflammation or hemorrhage. Dogs can recover spontane-
ously from the acute phase within 2 to 4 weeks, after which
time they may eliminate the infection or remain subclinically
infected. Sequestration of organisms within the spleen may
occur, and the organisms may evade the host immune system
through antigenic variation.5 This subclinical phase may persist
for months to years.
Chronic CME develops in only some infected dogs. Factors
that influence the development of chronic disease are unclear, but
genetics may play a role. The presence of pancytopenia typifies
the severe chronic form of ehrlichiosis, and results from hypo-
plasia of all bone marrow cells.22 Clinical signs range in severity
and include lethargy, inappetence, bleeding tendencies, muco-
sal pallor, fever, weight loss, lymphadenopathy, splenomegaly,
dyspnea, anterior uveitis, retinal hemorrhage and detachment,
polyuria/polydipsia, and edema.15,22-24 Polymyositis occurs in
some dogs, which can be manifested by diffuse muscle wasting
and tetraparesis.25 Secondary opportunistic infections such as
viral papillomatosis, protozoal infections, and bacterial urinary
tract infections can also develop, although the precise underly-
ing mechanism of immunosuppression, and how it relates to
successful persistence of E. canis, has not yet been elucidated
(see Figure 26-5).26,27 Marked granular lymphocytosis and bone
marrow plasmacytosis may occur, sometimes accompanied by a
monoclonal gammopathy, which may lead to misdiagnosis of
lymphocytic leukemia or multiple myeloma, respectively. This
has led to the recommendation that all dogs with well-differ-
entiated lymphocytosis or otherwise unexplained monoclonal
gammopathy be tested for E. canis infection.28 Protein-losing
nephropathy may develop as a result of immune-complex
glomerulonephritis.
FIGURE 28-3  Retinal hemorrhage and detachment in a 4-year old female spayed
Rhodesian ridgeback mix with canine monocytic ehrlichiosis. The dog also had mod-
erate to severe, poorly regenerative, macrocytic hypochromic anemia (16.4% with a
reticulocyte count of 49,000), lymphopenia (94 cells/µL), thrombocytopenia (26,000
platelets/µL), and large numbers of circulating nucleated red blood cells (75/100 WBC).
Systolic blood pressure was within normal limits. (Courtesy of University of California,
Davis Veterinary Ophthalmology Service.)
In reports of feline ehrlichiosis, clinical and laboratory find-
ings have generally been similar to those in dogs; one cat had
polyarthritis.16
Physical Examination Findings
Common physical examination findings in dogs with CME are
lethargy, fever, peripheral lymphadenopathy, and splenomegaly.
Ocular and nasal discharge, mucosal petechial hemorrhages, epi-
staxis, peripheral edema, and/or neurologic signs may be evident.
Ocular abnormalities include anterior uveitis, hyphema, retinal
hemorrhage, retinal detachment, and optic neuritis, with ante-
rior uveitis being most common (Figure 28-3).24,29 Neurologic
signs include twitching, ataxia, seizures, vestibular signs, hyper-
esthesia, and cranial nerve defects. Dogs with chronic ehrlichio-
sis may have thin body condition or diffuse muscle atrophy and
mucosal pallor. Findings in cats with ehrlichiosis have been simi-
lar to those in dogs, and include lethargy, splenomegaly, lymph-
adenopathy, petechial hemorrhages, and retinal detachment.16,17
Diagnosis
Laboratory Abnormalities
Complete Blood Count
Thrombocytopenia and occasionally mild leukopenia and a
nonregenerative anemia occur 1 to 4 weeks after infection with
E. canis. Mild thrombocytopenia, with increased mean platelet
volume, may persist during the subclinical phase. Classically,
dogs with chronic CME are pancytopenic, but more commonly,
nonregenerative anemia and thrombocytopenia are noted. In
some dogs, regenerative anemia or leukocytosis due to a neutro-
philia and band neutrophils are present. Lymphopenia occurs
in most affected dogs, but in some dogs, moderate to marked
granular lymphocytosis (up to 17,000/µL) can occur. Normo-
blastosis, that can exceed 50 nucleated RBC per 100 WBC, may
be present.

In some dogs, morulae are visualized within circulating
monocytes (Figure 28-2). The finding of morulae within mono-
cytes using cytologic evaluation of blood smears is insensitive,
especially in dogs with chronic infection, and does not distin-
guish between E. canis and E. chaffeensis infection. Use of buffy
coat smears, thin smears of blood collected from the margin of
the pinna, or splenic aspirates increases the sensitivity for detec-
tion of morulae. In one study, after careful searching, morulae
were found in only 2 of 19 dogs with CME.22
Serum Biochemical Tests
Serum chemistry abnormalities in chronic ehrlichiosis include
variable hypoalbuminemia, hyperglobulinemia, and elevated
ALT and ALP activities. Most often the hyperglobulinemia is
due to a polyclonal gammopathy.15 Monoclonal gammopathy
can also develop. Less commonly, increases in serum urea nitro-
gen and creatinine concentrations are present.22
Urinalysis
Transient proteinuria, with urine protein:creatinine ratios that
exceed 20 (reference range, <1) have been reported in dogs with
acute CME. This can resolve by 6 weeks after infection.30,31 Dogs
with chronic CME may also have evidence of proteinuria. Pyuria,
hematuria, and cylindruria may also be present.
Coagulation Profile
In addition to thrombocytopenia, coagulation abnormalities
in dogs with CME include prolongation of the buccal mucosal
bleeding time (BMBT), decreased platelet aggregation, and pro-
longation of the APTT.32,33
Cerebrospinal Fluid Analysis
Dogs with central nervous system (CNS) involvement may have
increased CSF protein concentrations and lymphocytic pleocy-
tosis.24 Although rarely found, morulae may be detected in cells
within the CSF.34
Bone Marrow Analysis
In dogs with chronic CME, bone marrow findings include hypo-
plasia or aplasia of all bone marrow elements, decreased iron
stores and marrow plasmacytosis (Figure 28-4). Bone marrow
mastocytosis has also been described.35 Myelofibrosis does not
typically develop in chronic CME.36 Some dogs have normal or
hypercellular marrows.
Diagnostic Imaging
Plain Radiography
Thoracic radiographs in dogs with CME often show no signifi-
cant abnormalities, but sometimes bronchointerstitial infiltrates
are present. This may reflect an underlying interstitial pneumonia.
Sonographic Findings
Findings on abdominal ultrasonography are nonspecific and
include splenomegaly, alterations in splenic echotexture,
enlargement and hypoechogenicity of the abdominal lymph
nodes, and scant peritoneal effusion. Increased renal echo-
genicity and decreased corticomedullary definition may occur in
dogs with glomerulonephritis.
Microbiologic Tests
Available diagnostic assays for ehrlichiosis in dogs and cats are
listed in Table 28-2.
CHAPTER 28  Ehrlichiosis 281
FIGURE 28-4  Bone marrow plasmacytosis in an 8-year-old female spayed Labrador
retriever with canine monocytic ehrlichiosis. Serum globulin was 12.1 g/dL (reference
range, 2.3-4.4 g/dL), and serum protein electrophoresis revealed a polyclonal gammopa-
thy. Plasma cells have a clock-faced nucleus and a clear area adjacent to the nucleus; two
plasma cells are identified with arrows. Mild to moderate megakaryocyte hyperplasia,
mild erythroid hypoplasia, and mild mature granular lymphocytosis were also present.
Romanowsky stain.
Serologic Diagnosis
Most often, the diagnosis of CME is made using serology, which
may be performed using indirect immunofluorescent antibody
(IFA) testing, ELISA technology, or Western blotting. Using IFA
testing, which is considered the gold standard, antibodies can be
detected between 7 and 28 days after initial infection. Dogs with
acute ehrlichiosis may have false-negative test results if sufficient
time has not elapsed for antibody production to occur. PCR
assays may be helpful for diagnosis in this situation. A positive
initial serum antibody titer may reflect previous exposure, and
not necessarily ehrlichial disease. Retesting should be performed
2 to 3 weeks later to demonstrate seroconversion, and results of
serology should be interpreted in light of a dog’s clinical signs
and the results of testing for other potential causes of the dog’s
illness. Dogs with chronic E. canis infection frequently have
extremely high IFA titers, sometimes greater than 1:600,000,
and these antibodies may persist in the face of treatment, sug-
gesting persistence of the organism.22 Seroconversion does not
generally occur in dogs with chronic disease, although antibody
titers may decline in some dogs with treatment. High titers do
not correlate with the severity of hyperglobulinemia, disease in
general, or duration of illness. Because of variability of report-
ing between laboratories, there is no standard “cutoff” titer
that is used to separate positive and negative results. Serologic
cross-reactivity to other Ehrlichia species occurs, which includes
E. ewingii and especially E. chaffeensis. Cross-reactivity to
A. phagocytophilum antigens can occur to a lesser extent. In
areas where other rickettsial agents are endemic, Western blot-
ting has been used in an attempt to confirm that positive anti-
body titers on IFA are truly to E. canis antigens. However,
Western blotting is laborious to perform and interpret and not
routinely available, and when E. canis antigens are the target
used, it may be difficult to distinguish between E. canis and E.
chaffeensis infection.37 As a result, Western blotting has pre-
dominantly been used on a research basis.
282 SECTION 2  Bacterial Diseases

TABLE 28-2
Diagnostic Assays Available for Ehrlichiosis in Dogs and Cats
Assay Specimen Type Target Performance
Cell culture Whole blood Ehrlichia canis; Not widely offered or utilized for routine diagnostic pur-
Ehrlichia ewingii poses. Requires several weeks’ incubation.
cannot be cultured
Morula detection Whole blood, buffy E. canis or Low sensitivity (especially for chronic E. canis infection).
coat smears, body E. ­ewingii E. canis morulae cannot be distinguished from those
fluids, ­tissue morulae of E. chaffeensis, and E. ewingii morulae cannot be
aspirates ­differentiated from those of A. phagocytophilum.
­Morulae may be confused with platelets, ­cytoplasmic
granules, phagocytized nuclear material, and
­lymphoglandular bodies.
IFA serology Serum Antibodies to Acute and convalescent serology is required for diagnosis
E. canis of acute infection, because initial results may be ­negative
in dogs with acute disease, and positive results can reflect
previous exposure rather than active infection. Dogs
with chronic infection generally do not seroconvert.
Cross-reactivity occurs to other ehrlichial species and
­occasionally to Anaplasma spp.
ELISA serology Serum Antibodies to E. Rapid, inexpensive, can be performed as an in-practice test.
canis or E. ewingii Similar limitations as for IFA. Lack of quantitation limits
antigens ability to document seroconversion.
Western Serum Antibodies to Technically difficult; primarily used on a research basis to
­immunoblotting specific E. canis or identify serologic responses to specific ehrlichial species.
E. ewingii antigens May be difficult to distinguish antibody responses to
E. canis and E. ewingii.
PCR Whole blood; spleen, E. canis, E. muris, or Confirms active infection. Sensitivity and specificity may
lymph node or bone E. ewingii DNA vary depending on assay design and specimen type.
marrow aspirates; ­Sensitivity for diagnosis of chronic CME may be low.
buffy coat or tissue Assays that specifically detect E. muris are not widely
specimens available on a commercial basis.

CME, Canine monocytic ehrlichiosis; IFA, immunofluorescent antibody.

A variety of ELISA assays have been developed for detec-
tion of antibodies to E. canis. A point-of-care lateral-flow
ELISA device for the simultaneous detection of canine heart-
worm antigen, antibodies to E. canis or E. ewingii, antibodies
to Borrelia burgdorferi, and antibodies to Anaplasma spp. in
canine serum, plasma, or whole blood has been marketed for
use in companion animals (SNAP 4Dx Plus, IDEXX Labora-
tories, Westbrook, ME), which includes recombinant surface
proteins of E. canis and E. ewingii on a single spot. Accord-
ing to the manufacturer, when IFA and Western blotting were
used as the gold standard, the sensitivity and specificity of the
E. canis antigen for detection of E. canis antibodies in 104
samples positive for E. canis antibodies and 236 samples nega-
tive for E. canis antibodies was found to be 96.2% and 100%,
respectively. Other point-of-care ELISA assays for detec-
tion of E. canis antibodies have also been developed. A sili-
con disc-based assay is available in the United States through
Antech Diagnostic laboratories that detects Dirofilaria immi-
tis antigen and antibodies to E. canis, Anaplasma spp., and
B. ­burgdorferi (Accuplex 4, Antech Diagnostics, Irvine, CA).
The performance of this assay for diagnosis of ehrlichiosis has
not been thoroughly investigated at the time of writing. The
incidental finding of E. canis seroreactivity in dogs screened
using these assays for heartworm antigenemia should prompt
performance of a thorough physical examination and basic
laboratory testing (CBC, chemistry panel, and urinalysis) to
evaluate for thrombocytopenia, hyperglobulinemia, and pro-
teinuria. When sick dogs test positive, quantitative serology
should be performed so that a titer can be obtained as a base-
line for acute and convalescent serologic testing or, for dogs
suspected to have chronic CME, to evaluate for titers of very
high magnitude that might be consistent with a dysregulated
immune response to the organism.
Molecular Diagnosis Using the Polymerase Chain Reaction
Whole-blood PCR assays for E. canis DNA is more sensitive for
early diagnosis of CME than IFA or ELISA in dogs with acute
disease. PCR assays are widely available for routine diagnosis of
E. canis infection. Several laboratories offer panels that include
PCR assays for a variety of different vector-borne pathogens.
The results of these assays should be interpreted in light of a
dog’s history, clinical signs, and the results of appropriate sero-
logic assays; the last should be performed to support the results
of PCR testing. PCR assays for E. canis may be performed on

blood, lymph node aspirates, splenic aspirates, or bone marrow.
Convalescent IFA or ELISA testing is much more sensitive than
PCR assays for diagnosis of chronic CME.22,38,39 The sensitivity
of PCR assays for diagnosis of CME when performed on bone
marrow in dogs with chronic ehrlichiosis can range from 25%
to 68%, depending on the laboratory.22 The use of PCR assays
in the absence of serology is currently not suitable for screening
potential blood donors for infection. PCR assays may be useful
to confirm infection in the first week of illness, when serologic
assays are often negative. Depending on the assay used, when
positive, PCR can also be used to confirm the Ehrlichia species
involved.
Blood Culture
E. canis can be cultured in certain cell lines, such as DH82
cells. This is time-consuming and generally performed only on
a research basis.
Pathologic Findings
Gross pathologic findings in CME include widespread pete-
chial and ecchymotic hemorrhages, generalized pallor, edema,
lymphadenopathy, and splenomegaly.40,41 Ascites may also be
present. On histopathology, lymphoid and plasma cell hyper-
plasia, lymphoplasmacytic infiltrates, and vasculitis may be
present in numerous organs, such as the brain, eye, spinal cord,
spleen, liver, kidneys, lymph nodes, bone marrow, and lungs.
Histiocytic infiltrates may be found in lymph nodes. With chro-
nicity, the proportion of plasma cells increases. Non-suppu-
rative meningitis and perivascular cuffing within the CNS can
also occur.40-42 Glomerulonephitis may be evident,40 although
dogs with acute CME have had interstitial nephritis and elec-
tron microscopic abnormalities that are more consistent with
minimal-change glomerulonephritis, with fusion of podocyte
processes.30 Little information is available in regard to the prev-
alence and type(s) of glomerulonephritis that develop in dogs
with chronic CME.
Treatment and Prognosis
Antimicrobial Treatment
The treatment of choice for CME is doxycycline (10 mg/kg PO
q24h) (Table 28-3). It was the consensus of the ACVIM Infec-
tious Disease Study Group that dogs and cats should be treated
for a minimum of 28 days.28 Mixed results have been obtained
in studies that have evaluated the efficacy of doxycycline for
treatment of E. canis infection. One study suggested that acute
infection can be eliminated after treatment for just 16 days.43
Another study that used ticks to infect dogs showed a failure
of doxycycline, when given for 14 days, to eliminate the organ-
ism from subclinically infected dogs.44 In another study, dogs
with acute and subclinical infections became negative by PCR
assay on blood after 28 days of doxycycline treatment, but dogs
with chronic infections remained intermittently positive. How-
ever, ticks still became PCR positive after they fed on treated
dogs, regardless of the stage of disease when treatment was
initiated.45
Whether or not persistence of infection occurs, most dogs
with acute disease show clinical improvement within 24 to 48
hours. Dogs with severe chronic disease may not respond to ther-
apy, or cytopenias may resolve over a period of several months.
Strong risk factors for mortality in one study were severe leu-
kopenia (WBC <930 cells/µL), severe anemia (HCT <11.5%),
CHAPTER 28  Ehrlichiosis 283
TABLE 28-3
Antimicrobial Drug Doses Used for Treatment of Ehrlichiosis
in Dogs and Cats
Interval Minimum
Drug Dose Route (hours) Duration (days)
Doxycycline 5 mg/kg PO, IV 12* E. canis 28 days;
E. ewingii
14 days
Oxytetracycline 7.5-10 IV 12 Until gastroin-
mg/kg testinal signs
abate, and then
change to oral
doxycycline as
above
*Or 10 mg/kg PO q24h.
hypokalemia (<3.7 mmol/L), and prolonged APTT (>18.25 s).46
Platelet counts generally improve and normalize by 2 weeks
following institution of therapy. After treatment, titers can
decline and become negative in 6 to 9 months. Some dogs
retain high titers for several years. Treatment for these dogs
should be based on resolution of platelet counts and improve-
ment of hyperglobulinemia, although hyperglobulinemia can
resolve over several months after treatment is discontinued.
The use of PCR assays on splenic aspirates could be considered
to determine if persistent infection is present in these dogs,
but whether ongoing treatment with doxycycline changes the
outcome for these dogs is unknown. Platelet counts should
be reassessed 1 and 3 months after discontinuation of ther-
apy, because of the potential for relapse or reinfection. Other
causes of illness (especially other vector-borne diseases such as
babesiosis or bartonellosis) should also be considered in dogs
that fail to respond to treatment.
Other drugs used to treat CME with variable success are
chloramphenicol, imidocarb dipropionate, and enrofloxa-
cin.47-50 Ehrlichia canis appears to have intrinsic gyrase-medi-
ated resistance to fluoroquinolones,51 so although their use can
be associated with clinical improvement, it is not recommended.
The antiprotozoal drug imidocarb dipropionate appeared effi-
cacious for treatment of E. canis infection in some studies but
not others.48,49
At this time, treatment of seroreactive but otherwise healthy
dogs that have normal routine bloodwork results is controver-
sial, because it is unknown if treatment changes outcome for
these dogs and has the potential to lead to antimicrobial resis-
tance or adverse effects of drug therapy.
Supportive Care
For dogs with CME that are dehydrated or anemic, IV flu-
ids or blood products may be required. Use of erythropoi-
etin and granulocyte colony-stimulating factor together with
­prednisone was associated with treatment success in a dog
with severe chronic ehrlichiosis in one case report.52 Des-
mopressin acetate (DDAVP) (1 mcg/kg SC q24h) for 3 days
appeared to be efficacious to treat bleeding disorders in a few
dogs with CME,33 and treatment resulted in reduction of the
BMBT and APTT. If thrombocytopenia fails to resolve with
284 SECTION 2  Bacterial Diseases
­ oxycycline administration, a short course (up to a week)
d sanguineus have been reported as a result of indiscriminate use
of therapy with immunosuppressive doses of glucocorticoids
could be ­considered in addition to ongoing therapy with
doxycycline.
Immunity and Vaccination
A vaccine for E. canis infection is currently not available,
but in one study vaccination of dogs with an attenuated
strain dramatically reduced disease in dogs challenged with
a virulent strain of E. canis.53 The immune response to E.
canis infection is not well understood. Because E. canis is an
intracellular pathogen, cell-mediated immune responses are
required for pathogen elimination. In one study, dogs with
acute infection developed CD8+ lymphocytosis that subsided
after several weeks, despite organism persistence.27 Cytokine
studies suggest that the immune response may vary with the
strain of E. canis involved.54,55 Genetic factors are also likely
to be important determinants of the immune response and
outcome of infection.
Prevention
Avoidance of tick-infested areas and routine inspection of dogs
for ticks after outdoor activities can help to prevent ehrlichio-
sis. Early removal of ticks may help to reduce transmission,
because of the 24- to 36-hour delay that occurs between tick
attachment and feeding. Clients should be instructed to remove
ticks properly and avoid handling ticks with bare hands or
crushing them, to prevent exposure to infected hemolymph. A
variety of devices are available to assist tick removal, which are
placed around the area where the mouthparts enter the skin to
avoid crushing or squeezing the tick and leaving the mouth-
parts behind. Fine-tipped tweezers can be used to grasp the tick
as close to the skin as possible, followed by steady retraction
to remove the tick. The bite wound should then be thoroughly
cleaned with a suitable antiseptic solution (such as iodine or
chlorhexidine-based antiseptics) or soap and water. Ticks can
be disposed in alcohol or tested for vector-borne pathogens
using PCR assays.
Topical ectoparasiticides with activity against ticks also
prevent tick-borne infectious diseases. Examples of canine
ectoparasiticides with activity against ticks include those that
contain amitraz, fipronil, pyrethroids (permethrin, etofenprox,
pyrethrin, deltamethrin, flumethrin), and selamectin. In one
field study, the use of monthly permethrin effectively prevented
infection with E. canis by kenneled dogs.56 Products that con-
tain pyrethroids and amitraz have the most potent activity
against ticks. Permethrin, deltamethrin, and amitraz cannot be
used in cats, and use of these products should be avoided on
dogs that co-habitate with cats. Flumethrin collars are avail-
able for use on cats (and on dogs) because unlike the other
pyrethroids, flumethrin does not require hepatic glucaronida-
tion for metabolism.57 Products that contain amitraz, a mono-
amine oxidase inhibitor, should not be used in dogs treated
with selective serotonin reuptake inhibitors (SSRIs) such as
fluoxetine, and pet owners that are receiving SSRIs should
also use alternative preventatives for their dogs. Preventatives
should be applied consistently at the recommended interval for
optimum activity. No preventatives completely protect dogs
or cats from tick attachment, especially where tick infestation
rates are high. Unfortunately, acaricide-resistant strains of R.
of these drugs.58
Low dose doxycycline (6.6 mg/kg q24h PO) has also been
used to prevent infection in dogs residing in kennels in which
E. canis infection is a problem. Resistance to doxycycline
remains a theoretical concern in this situation.
Because of chronic, subclinical infection, blood donor dogs
should be screened with serology (or serology and PCR assays)
for evidence of E. canis exposure or infection. All seropositive
dogs should be excluded as donors. Transport of chronically
infected dogs to non-endemic regions has the potential to intro-
duce the infection or new strains of E. canis to these regions if
the appropriate tick vectors are present.
Public Health Aspects
E. canis DNA has been detected in some human patients with
clinical signs of human monocytic ehrlichiosis,59 suggesting that
E. canis might be a cause of monocytic ehrlichiosis in people.
Appropriate precautions should be taken to prevent transmis-
sion when handling engorged ticks as well as blood and tissue
specimens from infected dogs, and care should be taken to pre-
vent needle-stick injuries.
Ehrlichia ewingii Infection
Etiology and Epidemiology
Ehrlichia ewingii is an unculturable bacterium that causes
granulocytic ehrlichiosis in humans and in dogs. It was first
recognized in dogs2 and occurs in North America and more
recently has been detected in dogs from Africa and Brazil.60,61
Infection occurs in the south-central and southeastern parts
of the United States, which reflects the distribution of the
primary tick vector, Amblyomma americanum. Transmis-
sion within the tick is transstadial. More than 40% of dogs
from an endemic area in Oklahoma and Arkansas were
seropositive.62 In another large study the overall seropreva-
lence was 14.5% in the central states (Oklahoma, Arkansas,
Missouri, and Kansas) and 5.9% in the southeast.63 In one
study, most cases occurred from May through July,64 but in
another, cases occurred throughout the year.65 In the study
from the south-central United States, dogs were more likely
to test positive with a PCR assay in August.62 E. ewingii is
maintained in white-tailed deer.66 The DNA of E. ewingii has
also been found in Dermacentor variabilis and R. sanguineus
ticks,67 but A. americanum is the only proven vector.
Clinical Features
Signs and Their Pathogenesis
In contrast to E. canis infection, E. ewingii infection appears
to cause only acute disease; a chronic phase of disease has not
been described. Like A. phagocytophilum, E. ewingii replicates
in neutrophils and delays neutrophil apoptosis, which prolongs
the life span of the host cell.68
Dogs with E. ewingii infection may show no signs, or fever,
lethargy, anorexia, and neutrophilic polyarthritis may occur.
After experimental infection, signs develop after an incubation
period of 3 to 4 weeks.69-71 Vomiting and diarrhea occur uncom-
monly. Neurologic signs have been described in some naturally
infected dogs,65 but the possibility of concurrent infections with

other pathogens such as Rickettsia rickettsii was not ruled out
in these dogs. E. ewingii can be found in apparently healthy
dogs, so dogs may act as a reservoir for infection.
Physical Examination Findings
On physical examination, dogs with E. ewingii infection may
have evidence of lethargy, fever, lameness, reluctance to move,
a stiff gait, joint effusion, and pain on joint palpation. Reported
neurologic signs include anisocoria, tremors, and a head tilt.65
Diagnosis
Laboratory Abnormalities
Common laboratory findings in dogs with E. ewingii infection
are nonregenerative anemia and thrombocytopenia.65,72 Reac-
tive lymphocytosis can also occur.65 The biochemistry panel
may be unremarkable or show mild nonspecific abnormali-
ties. Synovial fluid analysis reveals neutrophilic polyarthritis.
Morulae may be detected within granulocytes in the periph-
eral blood or synovial fluid, but are indistinguishable from
those of A. phagocytophilum. Nevertheless, the finding of
morulae in granulocytes in E. ewingii endemic areas and where
A. phagocytophilum infection is uncommon or absent is
strongly suggestive of E. ewingii infection. In experimental
infections, morulae are visible in the peripheral blood of some
dogs before the onset of clinical signs, around 2 to 3 weeks
postinfection.71
Microbiologic Tests
Serologic Diagnosis
Serologic diagnosis of E. ewingii infection is currently limited
to a point-of-care ELISA assay that detects the presence of
antibodies to a specific peptide of E. ewingii (SNAP 4Dx Plus,
IDEXX Laboratories, Westbrook, ME). Because this peptide is
combined on a spot with a peptide that detects antibodies to
E. canis, the antibody response to these pathogens cannot
be distinguished from one another. Cross-reactions may also
occur to other ehrlichial species such as E. chaffeensis. Anti-
bodies appear approximately 1 month after infection.71 Dogs
with acute illness may initially test negative with this assay,
and positive test results may reflect the presence of antibodies
from previous exposure or subclinical infection. A change from
a negative to a positive result over a 2- to 4-week time period
may help support a diagnosis of E. ewingii infection in endemic
areas.
Molecular Diagnosis Using the Polymerase Chain Reaction
Whole-blood PCR assays are available for specific diagnosis of
E. ewingii infection through some veterinary diagnostic labora-
tories. The sensitivity of these assays may vary between labo-
ratories. Currently E. ewingii–specific PCR assays are the only
means to confirm active infection with E. ewingii (as opposed
to infection with A. phagocytophilum or other Ehrlichia spe-
cies). In experimental studies, PCR becomes positive as early as
4 days after inoculation.71
Treatment and Prognosis
Antimicrobial Treatment
For E. ewingii infection, treatment with doxycycline results
in rapid (within 24 to 48 hours) clinical improvement. Treat-
ment for 2 to 4 weeks may be sufficient to eliminate infection.
CHAPTER 28  Ehrlichiosis 285
Subclinically infected dogs that test positive with whole-blood
PCR assays may spontaneously clear infection within weeks to
months. Treatment may not be required for these dogs.
Prevention
Prevention of E. ewingii infection involves avoidance of tick
exposure and use of tick preventatives (see previously). Early tick
removal also has the potential to reduce transmission. Poten-
tial blood donor dogs could be screened for infection with E.
ewingii–specific ELISA and PCR assays. PCR assays alone (i.e.,
without serology) should not be used to screen blood donors.
Public Health Aspects
Human infection with E. ewingii has been rarely described in
endemic regions of the United States.73 Affected people have
had headache, fever, and thrombocytopenia, with or without
leukopenia, and responded to doxycycline treatment. Most
were receiving immunosuppressive drug therapy. Although
direct dog-to-human transmission does not occur, blood from
affected dogs should be handled with caution.
Ehrlichia chaffeensis Infection
Ehrlichia chaffeensis causes human monocytic ehrlichiosis in
North America, an emerging disease that is characterized in
human patients by fever, headache, myalgia, thrombocytopenia
and leukopenia, and elevations in hepatic transaminases.74 Gas-
trointestinal signs, neurologic involvement, and a toxic shock–
like syndrome also occur in some infected people. In the United
States, it occurs primarily in the south-central, southeastern,
and mid-Atlantic states, which reflects the distribution of
A. americanum and the concurrent presence of white-tailed
deer, which are reservoirs for the organism (as for E. ­ewingii).
Evidence of E. chaffeensis DNA has also been found in
humans, dogs, other animal species (including cats18), and
ticks in Africa, Israel, Central and South America, and Asia.
The organism is transmitted transstadially within the tick,75
which feeds aggressively on humans. In naturally infected dogs,
E. ­chaffeensis infection has been associated with clinical signs
of lymphadenopathy, anterior uveitis, and epistaxis,76 but the
clinical implications of this infection for dogs are still unclear.
Dogs maintain high antibody titers and are PCR positive for
months after infection, which supports a possible role of the dog
as a reservoir.77
Ehrlichia muris-like Infection
Ehrlichia muris was first described in a wild mouse from Japan
in the mid-1990s78,79 and E. muris DNA was detected in a
febrile person in Russia for the first time in 2008.80 In 2009,
the DNA with strong homology to that of Ehrlichia muris was
detected in four humans from Minnesota and Wisconsin.80
These individuals had clinical signs and laboratory abnormali-
ties that resembled those of granulocytic anaplasmosis (see
Chapter 29), were seronegative to A. phagocytophilum, and
showed variable seroreactivity to E. chaffeensis. Two of the
individuals were solid-organ transplant recipients and the other
two people were apparently immunocompetent. Morulae were
not seen, but in mice E. muris forms morulae in monocytes.78
In 2012, DNA with homology to E. muris was detected in an
286 SECTION 2  Bacterial Diseases
ill dog from northern Minnesota that was seronegative for E.
canis and seroreactive to A. phagocytophilum,4 although the
extent to which this organism contributed to the dog’s clinical
signs was unclear. Because of the geographic distribution of
infection, Ixodes scapularis is the suspected tick vector (see
Chapter 29). E. muris DNA has been detected in I. scapularis
CASE EXAMPLE
Signalment: “Ditto” a 13-year old male neutered border collie
mix from Dixon, CA
History: Ditto was brought to the University of California, Davis,
because of a 4-day history of lethargy and inappetence.
Ditto had been drinking and there was no vomiting or
diarrhea. There was no history of exposure to ticks, toxins,
or trauma. Ditto was mainly confined to the backyard and
did not have access to standing water. The dog had been
adopted in Guam and brought to the United States several
years ago. There was no other significant travel history.
Ditto’s diet consisted of a commercial senior dry dog food.
Current Medications: Prednisolone (0.45 mg/kg PO q48h) for
allergic skin disease; this had been administered over the
preceding 10 days.
Physical Examination:
Body Weight: 22.3 kg
General: Quiet, reluctant to stand. T = 102.2°F (39.0°C), HR = 152
beats/min, panting, mucous membranes pale pink, CRT <2 s.
Approximately 5% to 7% dehydrated.
Integument: A sparse hair coat was present; there were no
other clinically significant abnormalities. No ectoparasites
were noted.
Eyes, Ears, Nose, and Throat: Severe dental calculus was
present.
Musculoskeletal: Body condition score was 6/9. The dog
would stand but was reluctant to walk.
Cardiovascular and Respiratory Systems: No clinically
significant findings were present.
Gastrointestinal and Urogenital Systems: Cranial
organomegaly was noted on abdominal palpation. Rectal
examination revealed no significant abnormalities.
Lymph Nodes: All peripheral lymph nodes were normal sized.
Laboratory Findings:
CBC:
HCT 28.0% (40%-55%)
MCV 72.4 fL (65-75 fL)
MCHC 30.4 g/dL (33-36 g/dL)
Reticulocyte count 7600 cells/µL (7000-65,000 cells/µL)
Nucleated RBC 1/100 WBC
WBC 19,600 cells/µL (6000-13,000 cells/µL)
Neutrophils 15,876 cells/µL (3000-10,500 cells/µL)
Lymphocytes 392 cells/µL (1000-4000 cells/µL)
Monocytes 1568 cells/µL (150-1200 cells/µL)
Eosinophils 392 cells/µL (0-1500 cells/µL)
Basophils 392 cells/µL (0-50 cells/µL)
Platelets 127,000 platelets/µL (150,000-400,000 platelets/µL)
MPV 20.5 fL (7-13 fL).
Serum Chemistry Profile:
Sodium 141 mmol/L (145-154 mmol/L)
Potassium 4.2 mmol/L (3.6-5.3 mmol/L)
ticks from northern Wisconsin.81 The extent to which E. muris
causes disease in dogs and humans in the United States and
other countries requires further investigation but it should be
considered as a possible cause of unexplained febrile illness
in dogs, whether or not they have evidence of antibodies to
E. canis.
Chloride 105 mmol/L (108-118 mmol/L)
Bicarbonate 18 mmol/L (16-26 mmol/L)
Phosphorus 6.1 mg/dL (3.0-6.2 mg/dL)
Calcium 8.6 mg/dL (9.7-11.5 mg/dl)
BUN 55 mg/dL (5-21 mg/dL)
Creatinine 1.9 mg/dL (0.3-1.2 mg/dL)
Glucose 66 mg/dL (64-123 mg/dL)
Total protein 6.1 g/dL (5.4-7.6 g/dL)
Albumin 2.2 g/dL (3.0-4.4 g/dL)
Globulin 3.9 g/dL (1.8-3.9 g/dL)
ALT 49 U/L (19-67 U/L)
AST 153 U/L (19-42 U/L)
ALP 98 U/L (21-170 U/L)
Creatine kinase 341 U/L (51-399 U/L)
Gamma GT 4 U/L (0-6 U/L)
Cholesterol 282 mg/dL (135-361 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL)
Magnesium 2.6 mg/dL (1.5-2.6 mg/dL).
Urinalysis: SGr 1.018; pH 7.0, protein 75 mg/dL, no bilirubin, no
glucose, hemoprotein 25 erythrocytes/µL, 0-2 WBC/HPF, 0-2
RBC/HPF, rare granular casts
Urine Protein: Creatinine Ratio: 19.3 (reference range, <1)
Plasma Antithrombin: 64% (reference range, 80%-120%)
Imaging Findings:
Thoracic Radiographs: The right cranial mainstem bronchus
was slightly widened throughout its visible length and
bronchiectasis was suspected. The cardiovascular and
remaining pulmonary structures were within normal limits
for the age of the patient.
Abdominal Radiographs: An ill-defined soft tissue structure
was present that displaced the caudal border of the
stomach cranially. The cecum was not well visualized.
The liver extended past the costochondral arches with
mildly rounded borders, which was interpreted as mild
hepatomegaly. The soft tissue structure was thought to
represent an enlarged lymph node, pancreas, or spleen.
Abdominal Ultrasound: The liver had normal parenchymal
architecture. The gallbladder contained hyperechoic
sludge. The spleen was enlarged with rounded edges and
a heterogenous, more hypoechoic parenchyma. Both
kidneys showed a somewhat thickened cortex with a good
distinction between cortex and medulla. The left renal pelvis
was mildly dilated, and the papilla was blunted. There were
several cystic lesions within the abdomen that measured up
to 3.6 cm × 5.4 cm. Septa were visualized within the cystic
lesions. The cystic lesions appeared to arise from lymph
nodes.
Cytologic Findings: Cytology of ultrasound-guided lymph
node aspirate: There was a moderate amorphous basophilic
proteinaceous background with low numbers of nucleated
cells and erythrocytes. Nucleated cells were composed
primarily of a mixed lymphocyte population with lower
numbers of macrophages and nondegenerate. neutrophils.

Microbiologic Testing: 4Dx SNAP test serology (IDEXX
Laboratories, ME): Positive for antibodies to Ehrlichia canis.
Weakly positive for antibodies to Anaplasma species.
Negative for antibodies to Borrelia burgdorferi and antigen
of Dirofilaria immitis.
Vector-borne disease serology (IFA): Positive for antibodies to
Ehrlichia canis at 1:163,840 and Anaplasma phagocytophilum
at 1:2560. Negative for antibody to Rickettsia rickettsii at <1:40.
Vector-borne real-time PCR panel (whole blood): Positive for
Ehrlichia canis DNA. Negative for Anaplasma phagocytophi-
lum, Anaplasma platys, Bartonella spp., Rickettsia spp., and
Borrelia burgdorferi DNA.
Diagnosis: Canine monocytic ehrlichiosis, characterized by
thrombocytopenia, abdominal lymphadenopathy, and
protein-losing nephropathy.
Treatment: Ditto was initially treated with IV crystalloids
(lactated Ringer’s solution supplemented with 20 mEq/L KCl),
famotidine (0.5 mg/kg IV q12h), and ampicillin (20 mg/kg IV
q8h). The hematocrit dropped to 18%, and 1 unit of packed
RBC was administered, which was followed by a transfusion
reaction, characterized by hemoglobinuria, vomiting, and
icterus. Systolic blood pressure remained within normal limits
throughout hospitalization. When the results of serology
for E. canis were obtained, antimicrobial drug treatment
was changed to doxycycline (5 mg/kg PO q12h). Enalapril
(0.25 mg/kg PO q24h) and aspirin (0.5 mg/kg PO q24h)
were used to manage the protein-losing nephropathy. Two
days after initiation of doxycycline treatment, the dog’s
attitude and appetite improved. At discharge (day 9 of
hospitalization), the hematocrit was 26.6%, reticulocytes
150,000 cells/µL, platelet count 496,000/µL, BUN 31 mg/dL,
creatinine 1.5 mg/dL, albumin 1.8 g/dL, globulin 5 g/dL, and
SUGGESTED READINGS
Komnenou AA, Mylonakis ME, Kouti V, et al. Ocular manifestations
of natural canine monocytic ehrlichiosis (Ehrlichia canis): a retrospec-
tive study of 90 cases. Vet Ophthalmol. 2007;10:137-142.
Mylonakis ME, Koutinas AF, Breitschwerdt EB, et al. Chronic canine
ehrlichiosis (Ehrlichia canis): a retrospective study of 19 natural
cases. J Am Anim Hosp Assoc. 2004;40:174-184.
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CHAPTER 28  Ehrlichiosis 287
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CHAPTER 29
Anaplasmosis
Jane E. Sykes and Janet E. Foley
Overview of Anaplasmosis
First Described: First reports of Anaplasma phagocytophi-
lum came from sheep in 1951 (Scotland).1 Canine infec-
tion was first reported in the United States (California) in
1982.2 Anaplasma platys was first reported in the United
States (Florida) in 1978.3
Cause: Anaplasma phagocytophilum (granulocytic anaplasmo-
sis), Anaplasma platys (thrombocytotropic anaplasmosis)
Affected Hosts: A. phagocytophilum causes disease in dogs,
cats, humans, ruminants (European strains), horses, and
camelids. A. platys causes thrombocytopenia in dogs.
Geographic Distribution: In the United States, A. phagocyto-
philum is most prevalent in the upper Midwest, northeast
and western states. Infection also occurs throughout con-
tinental Europe and the United Kingdom, Asia, and Russia.
The organism has been detected in dogs from Africa and
South America. A. platys occurs throughout the Americas,
Europe, Asia, Australia, the Middle East, and Africa.
Mode of Transmission: Tick vectors, primarily Ixodes ricinus-
persulcatus complex ticks transmit A. phagocytophilum.
Although unconfirmed, Rhipicephalus sanguineus is sus-
pected to be the major vector of A. platys.
Major Clinical Signs: The major clinical signs of A. phagocy-
tophilum infection are fever, lethargy, inappetence, and
lameness due to polyarthritis, although vomiting, diar-
rhea, cough, and neck pain may occur. A. platys usually
causes no signs, but fever and lethargy are possible.
Differential Diagnoses: Major differential diagnoses include
other tick-borne diseases (such as the ehrlichioses, rick-
ettsioses, Lyme borreliosis and babesiosis), bartonellosis,
leptospirosis, primary immune-mediated disease, and
lymphoma.
Human Health Significance: A. phagocytophilum causes
human granulocytic anaplasmosis. Dogs act as a sentinel
for human infection and may carry unfed ticks to humans
on their coats.
C
anine anaplasmosis is caused by Anaplasma
­phagocytophilum (formerly Ehrlichia equi, Ehrlichia
phagocytophila, and, in humans, the human granulo-
cytic ehrlichiosis [HGE] agent) and Anaplasma platys. These are
tick-borne, gram-negative, obligately intracellular bacteria that
belong to the family Anaplasmataceae. A. phagocytophilum
predominantly infects neutrophils but also eosinophils, where
290
it forms host membrane-enclosed morulae. Ticks that belong to
the Ixodes ricinus-persulcatus complex are the major vectors for
A. phagocytophilum. Anaplasma platys forms morulae within
platelets. The vector of A. platys is probably Rhipicephalus
­sanguineus. As with the ehrlichioses, co-infections with other
pathogens that are transmitted by the same or other tick species
may occur and influence the clinical manifestations of disease.
Anaplasma phagocytophilum Infection
Etiology and Epidemiology
A. phagocytophilum causes granulocytic anaplasmosis in dogs,
humans, horses, and, in Europe, domestic ruminants that
include sheep, cattle, goats, and deer. Cats and camelids may
also be affected. A variety of wild animal species, which include
rodents and deer, act as reservoir hosts. For the midwestern and
eastern United States, white-footed mice (Peromyscus leuco-
pus) and eastern chipmunks may act as reservoirs, whereas in
the western states, dusky-footed woodrats, gray squirrels, and
chipmunks have been implicated. In Europe, bank voles, wood
mice, shrews, and deer are likely reservoirs. Many strains of
A. phagocytophilum exist that differ in pathogenicity and host
tropism. For example, strains that infect domestic ruminants in
Europe and white-tailed deer in the United States appear to be
distinct from those that infect horses, humans, and dogs. Dogs,
cats, and humans are accidental hosts and are not important in
the transmission of infection to other host species.
The geographic distribution of the disease follows that of the
tick vectors (Figure 29-1). In North America, the tick vectors of
A. phagocytophilum are Ixodes scapularis in the northeastern
and upper midwestern states, and Ixodes pacificus in the West.
In Europe, the primary vector is Ixodes ricinus, and the disease
has been described in dogs throughout continental Europe and
in the UK. Ixodes persulcatus and Dermacentor silvarum ticks
transmit the organism in Asia and Russia. Other Ixodes spp.
ticks have also been implicated in transmission. Evidence of
A. phagocytophilum infection has been found in dogs from
Brazil and Tunisia, and a closely related organism was found
in dogs from South Africa.4-6 In humans, rare reports exist of
direct transmission that followed close contact with blood or
respiratory secretions, transplacental spread, or transmission
through blood transfusion.
Many seroprevalence studies of dogs have been reported
worldwide.7 The seroprevalence varies with geographic loca-
tion and whether the dogs studied were sick or healthy. The
prevalence of positive antibody titers in dogs from some regions
of Europe and North America exceeds 50%. A study that used a
commercially available ELISA assay to determine the prevalence

FIGURE 29-1  Distribution of Ixodes scapularis and Ixodes pacificus, the vectors of
Anaplasma phagocytophilum, in the United States. The distribution of granulocytic ana-
plasmosis follows that of the tick vectors.
of seroreactivity to A. phagocytophilum in more than 400,000
dogs from the United States revealed wide variation in seroprev-
alences. Some counties in the upper Midwest and northeastern
United States had seroprevalences that exceeded 40%, although
the overall seroprevalence in these regions were 6.7% and 5.5%,
respectively.8 Serologic cross-reactivity with A. platys may also
influence these data.
The seasonal pattern of disease reflects times of peak nymphal
and adult tick activities, as well as periods when humans and
their dogs are active outdoors. In the western United States,
A. phagocytophilum infection occurs most frequently in dogs
between April and July, when nymphal ticks are abundant.
Some infections occur in October, during early questing of adult
ticks. In Minnesota and Wisconsin, most cases are diagnosed
in late spring (May, June) and fall (October, November).9-11
In a study from Berlin, most cases occurred between April and
­September.12 The median age of clinically affected dogs is 6 to 8
years (range, 6 months to 14 years).9,10,12-14 In the upper Mid-
west of the United States, a bimodal age distribution has been
recognized, with 25% of dogs 1 year of age or less, and 50% of
dogs at least 8 years of age.10 Affected cats in the northeastern
United States have ranged from 4 months to 13 years of age
(mean, 3.7 years).15 Although no breed predispositions are rec-
ognized, in one study, golden retrievers comprised almost half
of affected dogs; this may reflect the popularity of these dogs for
outdoor activities.14 Infection with other tick-borne pathogens
is a risk factor for A. phagocytophilum infection. Co-infection
with Borrelia burgdorferi, the cause of Lyme disease, is com-
mon because B. burgdorferi is transmitted by the same Ixodes
tick species.8,10,11 In northern California, dogs seroreacted to
A. phagocytophilum were 18 times more likely to be seroposi-
tive for Bartonella vinsonii subspecies berkhoffii than dogs that
were seronegative.16
CHAPTER 29  Anaplasmosis 291
FIGURE 29-2  Life cycle of Ixodes scapularis ticks and Anaplasma phagocytophilum
infection. Uninfected larvae (top right) hatch in the late spring and acquire infection from
small rodents in the summer. They then over-winter (often in protected mouse burrows)
and then molt into nymphs the following spring. The nymphs feed in the late spring or
early summer on a variety of animal species including rodents, humans, deer, and dogs.
Nymphs molt into adults in the late summer to fall, which subsequently feed on large
mammals such as deer, where they mate and drop off. The females then lay eggs and die.
Dogs, cats and humans become infected by nymphs or adult ticks.
Clinical Features
Signs and Their Pathogenesis
A. phagocytophilum is transmitted transstadially within the
tick (i.e., from larva to nymph to adult), and not transovarially
(from adult to egg). Dogs and cats thus become infected after
exposure to infected nymphs or adult ticks, which acquire infec-
tion when they feed on wild animal reservoir hosts as larvae
or nymphs (Figure 29-2). Ticks must attach for 36 to 48 hours
for transmission to occur. Once A. phagocytophilum enters the
bloodstream, it attaches to the sialylated ligands on the surface
of neutrophils, such as P-selectin glycoprotein ligand-1.7 The
organism then enters the neutrophil through caveolae-mediated
endocytosis. A. phagocytophilum survives in the harsh neutro-
phil environment through dysregulation of neutrophil function
and by bypassing phagolysosomal pathways. It inhibits neutro-
phil superoxide production and can reduce neutrophil motility
and phagocytosis. A. phagocytophilum also reduces neutrophil
adherence to endothelium and inhibits neutrophil transmigra-
tion into tissues, possibly through downregulation of selectin
molecule expression.7 This may promote its survival in periph-
eral blood.
Normally, neutrophils circulate for 10 to 12 hours before
they enter tissues and undergo death through apoptosis.
A. phagocytophilum delays neutrophil apoptosis, which allows
it to survive longer periods of time within the neutrophil.7
A. phagocytophilum may also infect other cell types, such as
bone marrow cells, endothelial cells, and megakaryocytes,
although the importance of these cells in the pathogenesis of
infection is not clear.
The clinical signs and laboratory abnormalities that occur
in dogs and cats with granulocytic anaplasmosis probably
vary somewhat in different geographic locations as a result of
local strain variation. The vast majority of dogs infected with
292 SECTION 2  Bacterial Diseases

A. phagocytophilum show no clinical signs. Some dogs and cats
develop a self-limiting febrile illness, which in dogs occurs after
an incubation period of 1 to 2 weeks. Lethargy occurs in almost
all clinically affected cats and dogs. Fever and inappetence are
also common findings. Lameness, reluctance to move, polydip-
sia, vomiting, diarrhea, and a soft cough can also occur. Gener-
alized lymphadenopathy and splenomegaly develop as a result
of reactive lymphoid hyperplasia and, in the spleen, concurrent
extramedullary hematopoiesis.17 Uncommonly, hemorrhage,
manifested as mucosal petechiae, melena, or epistaxis, has been
reported in naturally infected dogs, but co-infections with other
tick-borne pathogens may contribute to these signs in some
dogs.9,12,13,18 Neurologic signs such as seizures, circling, cervi-
cal pain, and decreased placing reactions have been described
in a few dogs from the upper Midwest with granulocytic ana-
plasmosis, but one dog with seizures had a history of idiopathic
epilepsy.9,18 Neurologic signs and detection of the organism in
the CSF have been uncommonly reported in humans.19
Infection with A. phagocytophilum results in mild to mod-
erate thrombocytopenia, although other cytopenias may also
occur (Table 29-1). The mechanism(s) of these hematologic
abnormalities remain unclear. Anti-platelet antibodies occur
in serum from humans and dogs with granulocytic anaplasmo-
sis,12,20 and so immune-mediated mechanisms may contribute to
thrombocytopenia. However, thrombocytopenia occurs in acute
disease, before antibodies are noted, so other mechanisms may
be important. The bone marrow of infected dogs shows mega-
karyocyte hyperplasia, so platelet destruction may be involved.21
Impaired neutrophil function as a result of A. ­phagocytophilum
infection may predispose to development of secondary opportu-
nistic infections or influence the outcome of co-infections with
other tick-borne pathogens such as B. burgdorferi. Although
uncommon, opportunistic infections have been occasionally
documented in humans and dogs with granulocytic anaplasmo-
sis and are well in small ruminants.22
Infection with A. phagocytophilum may be self-limiting in
dogs and cats, with minimal fatality or chronic disease mani-
festations. The extent to which A. phagocytophilum can persist
in tissues and contribute to chronic disease manifestations in
humans and dogs has been controversial, and may be dependent
on the infecting strain and host immune response to infection.
In one study, treatment of dogs that had been experimentally
inoculated with A. phagocytophilum with prednisolone up to
6 months after infection was followed by the development of
positive PCR results for the organism and, in some dogs, throm-
bocytopenia and reappearance of morulae on blood smears.23
One dog infected with a California strain of A. phagocytophi-
lum was persistently PCR-positive through day 60 postinfec-
tion, the last time point evaluated.23
Physical Examination Findings
The most common findings on physical examination in dogs
include lethargy, fever (up to 106.7°F [41.5°C]), dehydration,
tachypnea, mild peripheral lymphadenopathy, and splenomeg-
aly.7,10 Scleral injection may be noted. Lameness, reluctance to
move, swollen joints, and pain on joint manipulation may also
be detected in some dogs. Increased lung sounds, abdominal
pain, epistaxis, petechial hemorrhages, and neurologic signs
such as circling, cervical pain, and decreased placing reactions
occur uncommonly.
Physical examination findings reported in cats have been
similar to those described in dogs, and include fever, leth-
argy, tachypnea, increased lung sounds, mild abdominal pain,
hepatomegaly, splenomegaly, vomiting, ataxia, hyperesthesia,
muscle and joint pain, lameness, conjunctivitis, and ocular
discharge.15,24,25
Diagnosis
Granulocytic anaplasmosis should be suspected in dogs and
cats with acute febrile illness and thrombocytopenia that reside
in endemic areas, regardless of tick exposure history. Diagno-
sis relies on detection of morulae within granulocytes, results
of acute and convalescent serology, or molecular testing using
PCR assays. The diagnostic criteria for confirmed human gran-
ulocytic anaplasmosis are clinical signs and laboratory find-
ings suggestive of granulocytic anaplasmosis together with (1)
detection of morulae within neutrophils combined with a single
positive reciprocal antibody titer to A. phagocytophilum of
at least 80; (2) a fourfold increase or decrease in the antibody

TABLE 29-1
Hematologic Abnormalities in Nine Dogs with Granulocytic Anaplasmosis in Northern California*
Number ­below Number within Number above Range for dogs
the Reference the Reference the Reference with Granulocytic
Test Reference Range Range Range Range Anaplasmosis
Hematocrit (%) 40-55 6 3 0 19-45
Neutrophils (cells/µL) 3000-10,500 0 6 3 3513-18,592
Band neutrophils (cells/µL) Rare 0 1 8 0-1282
Monocytes (cells/µL) 150-1200 1 7 1 142-1346
Lymphocytes (cells/µL) 1,000-4000 7 2 0 71-2693
Eosinophils (cells/µL) 0-1500 0 9 0 0-328
Platelets (cells/µL)† 150,000-400,000 6 2 0 37,000-262,000

*Diagnosis was based on compatible clinical signs and either morulae within circulating neutrophils and/or a positive real-time PCR assay result for
A. phagocytophilum.
†Platelets were clumped for one dog.
 CHAPTER 29  Anaplasmosis 293

titer within 4 weeks; (3) a positive PCR test result using specific
A. phagocytophilum primers; or (4) isolation of A. ­phagocytophilum
from blood.22 These criteria could also be applied to dogs. The use
of multiple diagnostic modalities may be needed to confirm the
diagnosis of granulocytic anaplasmosis in some dogs.
Laboratory Abnormalities
Complete Blood Count
Thrombocytopenia occurs in approximately 90% of dogs with
granulocytic anaplasmosis.9,10,12,13 The platelet count in throm-
bocytopenic dogs may occasionally be as low as 5000 platelets/
µL, although more often it is mild to moderately decreased (see
Table 29-1).9,10,12-14 The majority of affected dogs are lympho-
penic, but lymphocytosis can occur.9,12,13 Circulating reactive
lymphocytes may be present. Anemia is common and typically
mild and nonregenerative. Both neutrophilia and neutropenia
occur, but most dogs have neutrophil counts that lie in the lower
half of the reference range. Low numbers of band neutrophils,
as well as mild neutrophil toxicity, are often present. Monocyto-
penia or monocytosis can occur.9,12,13 Cytologic examination of
blood smears often reveals morulae within granulocytes (Figure
29-3). Morulae were detected in neutrophils from 36%, 56%,
67%, and 100% of dogs in four respective case series.9,12-14
Morulae appear as early as 4 days after experimental inocula-
tion of dogs and persist for 4 to 8 days.17 The morulae are indis-
tinguishable from those of Ehrlichia ewingii, so serology or PCR
assays are needed to confirm A. phagocytophilum infection.
In contrast to dogs, thrombocytopenia appears uncommon in
cats infected with A. phagocytophilum. None of 15 sick, PCR-
positive cats from the northeastern United States were throm-
bocytopenic.15 The most common hematologic abnormality
in cats has been lymphopenia. Morulae have been detected in
some cats with granulocytic anaplasmosis.
Serum Biochemical Tests
The most frequent serum biochemistry finding in dogs with
granulocytic anaplasmosis is mild to moderate hypoalbumin-
emia (Table 29-2). Mild hyperglobulinemia, mild electrolyte
abnormalities (hypokalemia, hyponatremia, and metabolic aci-
dosis), and a mild increase in the activities of serum ALP and to
a lesser extent ALT may occur.9,10,13,14
Urinalysis
Urinalysis in dogs with granulocytic anaplasmosis may reveal
isosthenuria, hyposthenuria, and proteinuria. Urine protein-to-
creatinine ratios in two affected dogs from the United States were

A B C
FIGURE 29-3  Intracytoplasmic morula (A) and cocci (B) of Anaplasma phagocytophilum (arrows) within neutrophils of an 8-year-old male neutered golden retriever with granulocytic
anaplasmosis; in (C), morulae are present in neutrophils within the synovial fluid of a 5-year-old curly-coated retriever that had polyarthritis.

TABLE 29-2
Findings on Serum Biochemistry Analysis in Nine Dogs with Granulocytic Anaplasmosis in Northern California*
Number below Number within Number above Range for dogs
Reference the Reference the Reference the Reference with Granulocytic
Test Range Range Range Range Anaplasmosis
Sodium (mmol/L) 145-154 4 5 0 142-147
Potassium (mmol/L) 4.1-5.3 2 7 0 3.3-4.9
Bicarbonate (mmol/L) 16-26 3 6 0 11-18
Albumin (g/dL) 2.9-4.2 6 3 0 1.5-3.4
Globulin (g/dL) 2.3-4.4 0 8 1 2.7-6.0
Total bilirubin (mg/dL) 0-0.4 0 8 1 0-1.0
Alanine aminotransferase (U/L) 19-70 0 8 1 26-122
Alkaline phosphatase (U/L) 15-127 0 7 2 26-653

*Diagnosis was based on compatible clinical signs and either morulae within circulating neutrophils and/or a positive real-time PCR assay result for
A. phagocytophilum.
294 SECTION 2  Bacterial Diseases

1.5 and 2.2,10 but there is no evidence that A. ­phagocytophilum
infection causes severe glomerulonephritis in dogs.
Synovial Fluid Analysis
Dogs with granulocytic anaplasmosis can develop neutrophilic
polyarthritis. Cytologic examination of synovial fluid in these
dogs reveals increased numbers of nondegenerate neutrophils.
In some affected dogs, morulae are found within these cells and
the synovial fluid can be PCR positive (Figure 29-3,C).
Diagnostic Imaging
Plain Radiography
Thoracic radiographs are usually normal in dogs with granu-
locytic anaplasmosis but may show a mild interstitial infiltrate.
Focal alveolar infiltrates occur as well.26
Sonographic Findings
Abdominal ultrasound may reveal splenomegaly, with a
hypoechoic spleen, or mild abdominal lymphadenopathy.
Microbiologic Tests
Diagnostic assays available for anaplasmosis in dogs and cats
are shown in Table 29-3.
Serologic Diagnosis
Diagnosis of granulocytic anaplasmosis can be accomplished
with acute and convalescent serology. Many veterinary laborato-
ries perform serologic testing using immunofluorescent antibody
(IFA) techniques. IgG antibodies are first detectable approximately
8 days after exposure, 2 to 5 days after morulae appear. As a
result, early in the course of illness, antibodies may be unde-
tectable, so PCR assays may be more useful for diagnosis of
acute infection in the absence of detectable morulae. Because
positive titers can reflect previous exposure, demonstration of
a fourfold rise in titer is required. Antibody titers may persist
for many months.14,23 In some human patients, antibody titers
have persisted as long as 3 years.22 In the United States, a silicon
disc-based ELISA assay is available commercially for detection
of antibodies to A. ­phagocytophilum (Accuplex 4, Antech Diag-
nostics, Irvine, CA). The performance of this assay when com-
pared with IFA requires further study. An in-clinic lateral-flow
ELISA device (IDEXX SNAP 4Dx Plus), which uses a recom-
binant Msp2/p44 protein, is also available for the detection of
antibodies to Anaplasma species in dog serum. Positive results
obtained with these ELISA assays do not imply that A. phago-
cytophilum is the cause of illness, and as with IFA testing, nega-
tive results can occur in dogs with acute illness due to the lag in
antibody production relative to the onset of clinical signs.
Serologic cross-reactivity between Anaplasma species occurs.
Dogs infected with A. platys also seroreact to A. ­phagocytophilum
antigen; this includes the recombinant Msp2 assay.8 Serologic
cross-reactivity between A. ­phagocytophilum and E. canis also
occurs, but is relatively uncommon and minor.13,26,27 Dogs diag-
nosed with granulocytic anaplasmosis that have antibodies to
A. ­phagocytophilum generally lack antibodies to E. canis using
IFA testing.7,10
Molecular Diagnosis Using the Polymerase Chain Reaction
Several conventional and real-time PCR assays for
A. ­phagocytophilum exist for detection of A. phagocytophilum

TABLE 29-3
Diagnostic Assays Available for Anaplasmosis in Dogs and Cats
Assay Specimen Type Target Performance
Cell culture Whole blood Anaplasma phagocytophilum; Not widely offered or utilized for routine diagnostic
Anaplasma platys cannot be purposes. Requires several weeks’ incubation.
cultured
Cytology Whole blood, buffy coat A. phagocytophilum or Low sensitivity (especially for A. platys infection).
smears, body fluids, A. platys morulae Morulae of A. phagocytophilum cannot be
­tissue aspirates ­distinguished from those of Ehrlichia ewingii.
Morulae may be confused with cytoplasmic
­granules or stain precipitate.
IFA serology Serum Antibodies to Anaplasma spp. Acute and convalescent serology is required for
diagnosis of acute infection, because initial results
may be negative in dogs with acute disease and
positive results may reflect previous exposure
rather than active infection. Cross-reactivity
­occurs between Anaplasma spp.
ELISA serology Serum Antibodies to Anaplasma spp. Rapid, inexpensive, can be performed as an in-practice
antigens test. Similar limitations as IFA. Lack of quantitation
limits ability to document seroconversion.
PCR Whole blood, splenic or A. phagocytophilum or Confirms active infection. Sensitivity and specificity
lymph node aspirates, A. platys DNA may vary depending on assay design and speci-
bone marrow aspirates, men type. Because healthy animals may be PCR
buffy coat specimens, positive, positive PCR results must be interpreted
tissue specimens in light of the clinical signs.

IFA, Immunofluorescent antibody.


DNA in peripheral blood, buffy coat, bone marrow, or splenic
tissue. These can be useful for early diagnosis of granulocytic
anaplasmosis in dogs and cats. The sensitivity and specificity
of PCR assays may vary depending on the assay design and
the laboratory used. Most, but not all, assays detect either
the 16S rRNA gene or the outer surface protein gene msp2
(p44). Assays that detect the msp2 gene are usually specific for
A. ­phagocytophilum, whereas assays that detect the 16S rRNA
gene may detect other Anaplasma species, and even other bacteria.
A. phagocytophilum DNA has occasionally been amplified from
healthy dogs, so results must be interpreted in light of the clini-
cal signs.11 In experimentally infected dogs, whole-blood PCR
becomes positive several days before and after morulae appear
on blood smears.17,23
Culture
A. phagocytophilum can be isolated in human promyelocytic
leukemia cell lines (HL-60) and tick embryo cell lines. Culture
is highly sensitive for diagnosis of acute infection in human
patients,28 but it is not routinely used in dogs for diagnostic
purposes because it requires special facilities, technical exper-
tise, and prolonged incubation.
Pathologic Findings
Little information is available regarding the pathology of gran-
ulocytic anaplasmosis in dogs. Tissue injury appears to result
from the host inflammatory response, rather than the bacterial
infection itself.29 In people, splenic lymphoid depletion, mac-
rophage aggregates and apoptosis within the liver, paracortical
lymphoid hyperplasia, and hemophagocytic cells within reticu-
loendothelial tissues have been described.30
Treatment and Prognosis
The treatment of choice for granulocytic anaplasmosis in dogs
is doxycycline (5 mg/kg PO q12h). The optimum duration
is unknown, but 2 weeks may be sufficient. The prognosis is
excellent. Most dogs show clinical improvement within 24 to
48 hours of treatment, although some dogs require more than
1 week of treatment before clinical signs resolve.10,13 Platelet
counts normalize 2 to 14 days after treatment is initiated.10 In
one study, 30% of 23 owners reported that their dog returned
to normal activities in 1 to 2 days after treatment was initiated,
30% reported this took 3 to 5 days, 21% 1 to 3 weeks, and
17% reported that it took a month or longer for their dogs to
return to normal activity.10 Death due to granulocytic anaplas-
mosis has not been described in dogs. Intravenous crystalloid
fluids and antiemetics may be required for supportive care until
clinical signs improve.
Immunity and Vaccination
The immune response to A. phagocytophilum infection is not
fully characterized. Both humoral and cell-mediated immune
responses appear to be important in clearance of infection. Host
cytokines such as IFN-γ may play a role in the initial control
of A. phagocytophilum infection31,32 but may also contribute to
the inflammatory process associated with disease.33 In lambs,
A. phagocytophilum evades the host immune response through
differential expression of MSP2, an outer surface protein
involved in immune recognition.34 Variation in MSP2 also occurs
in chronically infected woodrats.35 Further research is required
CHAPTER 29  Anaplasmosis 295
to document whether persistence occurs through similar mecha-
nisms in other host species. It is possible that recovery from nat-
ural infection confers long-term protection against development
of disease. Reinfection has not been reported in dogs, but was
described in one human patient.36 A vaccine for granulocytic
anaplasmosis is not available for companion animals.
Prevention
Infection may be prevented by avoidance of tick exposure,
prompt tick removal, and use of topical ectoparasiticides (see
Chapter 28). Although not a guaranteed protection, combina-
tions of topical imidacloprid and permethrin, or fipronil, amitraz
and (S)-methoprene, prevent transmission of A. phagocytophi-
lum to dogs from infected ticks.37,37a
Public Health Aspects
A. phagocytophilum infection was first recognized in humans
in the upper Midwest of the United States in the early 1990s
and has since been increasingly recognized in humans from the
United States, Europe, and Asia. Human granulocytic anaplas-
mosis closely resembles the disease in dogs, which has been
described as an “influenza-like illness after a tick bite.”38 Men
are affected slightly more frequently than women. In Europe,
the disease is most commonly reported in from Sweden and
Slovenia, where it was first reported in 1997.39 Morulae are
observed less often in humans with granulocytic anaplasmosis
in Europe, and the disease may be milder than that described in
humans from the United States.
The most common clinical signs reported in human patients
are myalgia, headache (which is often severe), malaise, and chills.
Anorexia, nausea, arthralgias, and cough may also occur.22,38,40
The disease typically resolves within 2 months in the absence of
appropriate antibiotic treatment. Occasionally more severe dis-
ease may occur. In one study, up to 17% of affected humans
required admission to an intensive care unit.38 Death occurs in
1% or fewer clinically affected humans, usually as a result of
complications such as a septic or toxic shock–like syndrome,
respiratory insufficiency, opportunistic fungal or viral infections,
rhabdomyolysis, acute renal failure, hemorrhage, or neurologic
disease.22 Severe illness tends to occur in humans of advanced age
or with concurrent immunosuppressive illness or drug therapy.
Laboratory testing of peripheral blood typically reveals normal or
slightly decreased white blood cell and platelet counts, sometimes
with a neutrophilic left shift, and mild to moderate elevation of
hepatic transaminase activities. In the United States, the disease
is reportable to the Centers for Disease Control and Prevention
(CDC). Dogs act as sentinels for human exposure and may be a
source of infection through mechanical carriage of infected ticks
to humans. Blood from affected dogs should be handled with
caution, and needle-stick injuries should be avoided.
Anaplasma platys Infection
Etiologic Agent and Epidemiology
Anaplasma platys infects and forms inclusions within platelets
and is the cause of canine cyclic thrombocytopenia, or thrombo-
cytotropic anaplasmosis. It does not appear to infect cats. The
organism is very widespread, and infections occur throughout
the Americas, Europe, Asia, Australia, the Middle East, and
296 SECTION 2  Bacterial Diseases
Africa. R. sanguineus ticks are believed to transmit A. platys,
because the DNA of A. platys has frequently been found in
R. sanguineus ticks worldwide, and dogs infected with A. platys
are often co-infected with E. canis. However, in a single attempt,
experimental transmission of A. platys by R. sanguineus ticks
failed.41 A. platys DNA has been found in other tick species,
such as Dermacentor auratus ticks in Thailand,42 ­Rhipicephalus
turanicus ticks from Israel,43 and Haemaphysalis spp. and
Ixodes nipponensis ticks from Korea.44 Different strains of
A. platys exist that appear to vary in pathogenicity. The organ-
ism has never been isolated in cell culture.
Clinical Features
Signs and Their Pathogenesis
Anaplasma platys causes thrombocytopenia in dogs, most often
in the absence of other clinical signs, although fever, lethargy,
lymphadenopathy, uveitis, pallor, and mucosal hemorrhages have
been described.45,46 Co-infections with other vector-borne patho-
gens may contribute to clinical signs in some dogs. Thrombocy-
topenia occurs 1 to 2 weeks after experimental inoculation. This
initial episode is associated with the highest number of organisms
in platelets, as detected using light microscopic examination of
stained blood smears.47 The platelet count nadir occurs 2 to 3
weeks postinfection and in some dogs may be lower than 20,000
platelets/µL. Visible organisms then disappear from platelets, and
the platelet count returns to normal or near-normal limits within
3 to 4 days. This also corresponds with a decrease in organism
load and failure to detect the organism in peripheral blood with
real-time PCR, although bone marrow and splenic aspirates may
remain positive.48 Cycles of thrombocytopenia and bacteremia
then occur at intervals of 7 to 14 days, after which infection and
thrombocytopenia persist in some dogs but morulae or inclusions
become more difficult to detect within platelets.47 The mecha-
nism of thrombocytopenia is unknown, but direct damage to
platelets, sequestration of platelets in the spleen, and immune-
mediated destruction have been hypothesized to play a role.
Co-infection of dogs with A. platys and E. canis may lead
to more severe anemia than occurs with isolated E. canis
infection.49 The duration of A. platys infection may also be
prolonged.
Physical Examination Findings
Most dogs with A. platys infection show no clinical signs. Fever,
petechial hemorrhages, and uveitis have been reported, but co-
infections with other vector-borne pathogens may have contrib-
uted to these signs.
Diagnosis
Laboratory Abnormalities
Usually, the only abnormality present on the CBC, biochemistry
panel, and urinalysis in dogs with A. platys infection is throm-
bocytopenia. A mild, nonregenerative anemia may be found.
The diagnosis can be made when morulae are seen within plate-
lets on blood smears, but this is insensitive, and other inclusions
or stain precipitate may be mistaken for organisms.
Microbiologic Tests
Direct Fluorescent Antibody
Direct fluorescent antibody techniques can be used to identify
A. platys in platelets, but these are not widely available for
routine diagnostic purposes. Molecular diagnostic assays have
overcome the need for direct fluorescent antibody or immuno-
cytochemical staining.
Serologic Diagnosis
Recent infection with an Anaplasma species can be detected
through the use of acute and convalescent serology with IFA.
Because antibodies to A. phagocytophilum bind to A. platys
antigens it is not possible to distinguish the immune response
to each of these pathogens with currently available assays. This
can be problematic in geographic regions where both A. platys
and A. phagocytophilum are found. A positive result on com-
mercially available ELISA assays also only indicates previous
exposure to an Anaplasma species. Dogs with recent infection
may be seronegative, because antibodies to A. platys are not
detectable for 1 to 2 weeks after inoculation. Because a single
positive test result only indicates previous exposure, and sero-
prevalence rates in endemic areas are high, thrombocytopenia
in a seropositive dog may be due to an etiology other than
­Anaplasma infection. Cross-reactivity to E. canis antigens does
not occur in dogs with A. platys infections, so positive titers to
E. canis and A. platys may represent previous exposure to both
pathogens, or to other Ehrlichia or Anaplasma species.
Molecular Diagnosis Using the Polymerase Chain Reaction
Several conventional and real-time PCR assays have been
described for detection of A. platys DNA. In the absence of
clearly identifiable morulae within platelets, molecular diag-
nostic testing with reliable PCR assays that specifically detect
A. platys DNA (as opposed to A. phagocytophilum DNA or
Anaplasma species DNA) is the only way to confirm active
infection. Real-time PCR assays that detect A. platys DNA
are available commercially and in combination with assays for
other vector-borne pathogens. Suitable specimens include whole
blood, buffy coats, or bone marrow and splenic aspirates,48
although it is not clear which of these specimens is optimal.
When buffy coats were used, PCR assays for A. platys became
positive as early 3 to 5 days after experimental infection, but
by day 21, negative results occurred in some dogs.48,49 The use
of splenic or bone marrow aspirates may yield positive results
when whole blood or buffy coat PCR for A. platys is negative
yet infection is still suspected.
Pathologic Findings
Pathologic findings have been described in a small number
of dogs that were experimentally infected with A. platys, and
necropsied early in the course of infection.50 The only gross
necropsy finding was generalized lymphadenomegaly. Lesions
consisted of reactive lymphoid hyperplasia and erythrophago-
cytosis by sinusoidal macrophages within lymph nodes and the
spleen, crescent-shaped regions of perifollicular hemorrhage in
the spleen, mild lymphoplasmacytic infiltrates in the renal inter-
stitium, and multifocal hyperplasia of Kupffer’s cells in the liver.
Megakaryocyte numbers in the bone marrow were increased in
some dogs.
Treatment and Prognosis
The recommended treatment for thrombocytopenic dogs
infected with A. platys is doxycycline. The optimum dose and
duration of treatment is unknown, but it is apparently eliminated
using regimens effective for treatment of E. canis infection.

Infection could not be detected with PCR for a 3-week follow-
up period after just 8 days of doxycycline treatment (10 mg/kg
PO q24h), which was initiated in the acute phase of infection.51
However, one dog remained infected after 2 weeks of treatment
with tetracycline. In another study, doxycycline treatment was
administered for 28 days, and infection could not be detected
after this time point, even after pharmacologic immunosuppres-
sion with dexamethasone several months later.49 Whether the
duration of infection at the time of treatment affects treatment
efficacy is unknown.
CASE EXAMPLE
Signalment: “Copper,” an 8-year old male neutered Labrador
retriever from Napa, CA
History: Copper was brought to the University of California,
Davis, VMTH emergency service for the problems of lethargy,
inappetence, apparent neck pain of 1 week’s duration, and
fever. The dog had been taken to a local veterinary clinic on
the day the illness began, and thrombocytopenia (113,000
platelets/µL) was detected on a CBC. ELISA serology for
antibodies to E. canis, B. burgdorferi, and A. phagocytophilum
and Dirofilaria immitis antigen (4Dx SNAP test, IDEXX
Laboratories) was negative. Treatment with enrofloxacin
(2.4 mg/kg PO q12h) and metronidazole was instituted,
during which time the dog improved clinically, but when
the metronidazole was discontinued after 5 days, lethargy,
inappetence, and signs of neck pain returned 2 days
later. A CBC at that time showed no clinically significant
abnormalities. Treatment with enrofloxacin was continued,
and methocarbamol (11.6 mg/kg PO q8h) and meloxicam
(0.1 mg/kg PO q24h) were instituted for the neck pain. The
owner took Copper’s rectal temperature at home on the
morning he was brought to the VMTH, and it was 105.6°F
(40.9°C). Copper lived on a 20-acre winery with a reservoir
and had access to wildlife and frequent tick exposure. There
had been no known exposure to toxins and no travel history.
The dog received monthly heartworm preventative, and flea
and tick preventative was used, but only when the owner
found ticks. He was normally fed a commercially available
dry dog food.
Current Medications: Enrofloxacin (2.4 mg/kg PO q12h),
methocarbamol (11.6 mg/kg PO q8h), and meloxicam
(0.1 mg/kg PO q24h).
Other Medical History: Nine months previously, Copper
was seen by the local veterinary clinic for neck pain. The
dog was treated with methocarbamol, a nonsteroidal
antiinflammatory drug, and rest, and the pain resolved after
7 days. The owners also reported that Copper had exhibited
some weakness of his left pelvic limb for several years.
Physical Examination:
Body weight: 43.1 kg
General: Quiet, alert, responsive, adequately hydrated.
T = 103.7°F (39.8°C), HR = 110 beats/min, panting, mucous
membranes pink, CRT <2 s.
Integument: No clinically significant abnormalities were
identified. No ectoparasites were noted.
CHAPTER 29  Anaplasmosis 297
Prevention
See the previous discussion of Anaplasma phagocytophilum
infection.
Public Health Aspects
Infection with A. platys has not been confirmed in humans.
Eyes, Ears, Nose, and Throat: Mild gingivitis and dental
calculus were present. No other significant abnormalities
were noted.
Musculoskeletal: Body condition score 4/9. The dog was
ambulatory but had a slightly stiff thoracic limb gait. A
decreased range of motion of coxofemoral joints was
detected, with the left worst than the right. There was no
evidence of joint pain or effusion.
Cardiovascular, Respiratory Systems, Gastrointestinal
and Urogenital Systems: No clinically significant findings
were identified. Rectal examination revealed no significant
abnormalities.
Lymph Nodes: All peripheral lymph nodes were normal sized
but slightly firm on palpation.
Neurologic Examination: Mentation and cranial nerve
function were within normal limits. The dog was reluctant to
move his neck on manipulation, especially to the right side,
and palpation of the cervical spine elicited pain. Decreased
placing reactions were noted in the left pelvic limb.
Laboratory Findings:
CBC:
HCT 35.8% (40%-55%)
MCV 64 fL (65-75 fL)
MCHC 35.8 g/dL (33-36 g/dL)
Reticulocyte count 10,200 cells/µL (7000-65,000 cells/µL)
WBC 7100 cells/µL (6000-13,000 cells/µL)
Neutrophils 6603 cells/µL (3000-10,500 cells/µL)
Band neutrophils 284 cells/µL
Lymphocytes 71 cells/µL (1000-4000 cells/µL)
Monocytes 142 cells/µL (150-1200 cells/µL)
Platelets 37,000/µL (150,000-400,000 platelets/µL)
MPV 17.2 fL (7-13 fL).
Slight toxicity was detected in neutrophils and band neutro-
phils, and multiple neutrophils contained basophilic intra-
cytoplasmic inclusions (morulae) (see Figure 29-3).
Serum Chemistry Profile:
Anion gap 23 mmol/L (10-24 mmol/L)
Sodium 145 mmol/L (145-154 mmol/L)
Potassium 3.7 mmol/L (3.6-5.3 mmol/L)
Chloride 115 mmol/L (108-118 mmol/L)
Bicarbonate 11 mmol/L (16-26 mmol/L)
Phosphorus 3.9 mg/dL (3.0-6.2 mg/dL)
Calcium 10.2 mg/dL (9.7-11.5 mg/dl)
BUN 12 mg/dL ( 5-21 mg/dL)
Creatinine 0.7 mg/dL (0.3-1.2 mg/dL)
Glucose 61 mg/dL (64-123 mg/dL)
Total protein 6.8 g/dL (5.4-7.6 g/dL)
298 SECTION 2  Bacterial Diseases
Albumin 3.1 g/dL (3.0-4.4 g/dL)
Globulin 3.7 g/dL (1.8-3.9 g/dL)
ALT 54 U/L (19-67 U/L)
AST 26 U/L (19-42 U/L)
ALP 106 U/L (21-170 U/L)
Creatine kinase 79 U/L (51-399 U/L)
GGT < 3 U/L (0-6 U/L)
Cholesterol 255 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL)
Magnesium 2.1 mg/dL (1.5-2.6 mg/dL).
Urinalysis: SGr 1.031; pH 8.0, 25 mg/dL protein, no bilirubin,
25 erythrocytes/µL hemoprotein, no glucose, 0-3 WBC/HPF,
15-25 RBC/HPF, no other significant abnormalities were
detected.
Imaging Findings:
Spinal Radiographs: Multiple right lateral projections of
the spine were reviewed. There were multiple sites of
mild ventral spondylosis deformans throughout the
thoracolumbar spine. Severe spondylosis was seen at the
lumbosacral space with sclerosis of the endplates either
side, most likely due to degenerative change. There was
severe osseous remodeling of the lumbar articular facets,
which was consistent with osteoarthrosis. Small areas of
intervertebral disc mineralization were seen at the L2-3 and
L4-5 spaces.
Thoracic Radiographs: The cardiovascular and pulmonary
structures were within normal limits. There was slightly
increased soft tissue opacity in the region of the sternal
lymph node on the right lateral projection.
Abdominal Ultrasound: The liver was mildly hypoechoic but
normal in size. The spleen was markedly enlarged but had
normal echogenicity. The rest of the abdominal organs were
within normal limits.
Microbiologic Testing: Aerobic bacterial urine culture: No
growth
ELISA serology for vector-borne pathogens (IDEXX 4Dx SNAP
test): Positive for antibodies to A. phagocytophilum. Nega-
tive for antibodies to E. canis and B. burgdorferi. Negative for
D. immitis antigen.
SUGGESTED READING
Carrade DD, Foley JE, Borjesson DL, et  al. Canine granulocytic ana-
plasmosis: a review. J Vet Intern Med. 2009;23:1129-1141.
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Treatment: Copper was treated with doxycycline (5 mg/
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17. Egenvall A, Bjoersdorff A, Lilliehook I, et al. Early manifestations
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20. Wong SJ, Thomas JA. Cytoplasmic, nuclear, and platelet autoan-
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21. Lilliehook I, Egenvall A, Tvedten HW. Hematopathology in dogs
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23. Egenvall A, Lilliehook I, Bjoersdorff A, et al. Detection of granulo-
cytic Ehrlichia species DNA by PCR in persistently infected dogs.
Vet Rec. 2000;146:186-190.
24. Heikkila HM, Bondarenko A, Mihalkov A, et  al. Anaplasma
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25. Tarello W. Microscopic and clinical evidence for Anaplasma
(Ehrlichia) phagocytophilum infection in Italian cats. Vet Rec.
2005;156:772-774.
26. Plier ML, Breitschwerdt EB, Hegarty BC, et al. Lack of evidence for
perinatal transmission of canine granulocytic anaplasmosis from a
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27. Breitschwerdt EB, Hegarty BC, Hancock SI. Sequential evaluation
of dogs naturally infected with Ehrlichia canis, Ehrlichia chaffeen-
sis, Ehrlichia equi, Ehrlichia ewingii, or Bartonella vinsonii. J Clin
Microbiol. 1998;36:2645-2651.
28. Aguero-Rosenfeld ME. Diagnosis of human granulocytic ehrlichio-
sis: state of the art. Vector Borne Zoonotic Dis. 2002;2:233-239.
29. Dumler JS, Barat NC, Barat CE, et  al. Human granulocytic
anaplasmosis and macrophage activation. Clin Infect Dis.
2007;45:199-204.
30. Lepidi H, Bunnell JE, Martin ME, et  al. Comparative pathol-
ogy, and immunohistology associated with clinical illness after
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2000;62:29-37.
31. Akkoyunlu M, Fikrig E. Gamma interferon dominates the murine
cytokine response to the agent of human granulocytic ehrlichiosis
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2000;68:1827-1833.
32. Birkner K, Steiner B, Rinkler C, et  al. The elimination of Ana-
plasma phagocytophilum requires CD4+ T cells, but is independent
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CHAPTER 29  Anaplasmosis 299
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interleukin-10 in a murine model of human granulocytic ehrlichio-
sis. Am J Pathol. 2001;158:1881-1888.
34. Granquist EG, Stuen S, Lundgren AM, et al. Outer membrane pro-
tein sequence variation in lambs experimentally infected with Ana-
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35. Rejmanek D, Foley P, Barbet A, et al. Antigen variability in Ana-
plasma phagocytophilum during chronic infection of a reservoir
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37a. McCall JW, Baker CF, Mather TN, et  al. The ability of a topi-
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42. Parola P, Cornet JP, Sanogo YO, et al. Detection of Ehrlichia spp.,
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43. Harrus S, Perlman-Avrahami A, Mumcuoglu KY, et al. Molecular
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Candidatus Midichloria mitochondrii and Babesia canis vogeli in
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1997;59:849-851.
CHAPTER 30
Rocky Mountain Spotted Fever
Linda Kidd and Edward B. Breitschwerdt
Overview of Rocky Mountain Spotted Fever
First Described: R. rickettsii was first described in Montana,
United States, 19091 (by Ricketts)
Cause: Rickettsia rickettsii, a gram-negative, obligately intra-
cellular bacteria
Affected Hosts: Humans and dogs
Geographic Distribution: North, Central, and South America
Primary Mode of Transmission: Ticks (Dermacentor spp.,
­Rhipicephalus sanguineus, Amblyomma spp.). Most affected
dogs lack a history of a tick bite.
Major Clinical Signs: Rocky Mountain spotted fever (RMSF)
is an acute, generally febrile illness. Clinical signs are con-
sistent with a vasculitis because R. rickettsii infects endo-
thelial cells. Major clinical signs include fever, vomiting,
ocular signs, lymphadenomegaly, splenomegaly, periph-
eral edema, cutaneous hyperemia and necrosis, polyar-
thritis, and neurologic signs. Signs may be complicated by
simultaneous infection with other tick-borne pathogens.
Differential Diagnoses: Infection with other tick-borne agents
such as A. phagocytophilum, Ehrlichia spp., ­Bartonella spp.,
Babesia spp., and Borrelia burgdorferi should be con-
sidered. Other causes of severe systemic decompensa-
tion and vasculitis, such as sepsis and SIRS due to other
causes may mimic RMSF. Leptospirosis and other causes
of vasculitis and thrombocytopenia may also mimic RMSF.
Other differential diagnoses for the neurologic signs seen
in RMSF must also be considered. Appropriate antimicro-
bial treatment with doxycycline must begin before the
diagnosis is confirmed by laboratory testing. Misdiagnosis
and delayed or inappropriate antimicrobial drug therapy
increase morbidity and mortality.
Human Health Significance: Owners and their physicians
should be contacted whenever RMSF is diagnosed in any
canine patient, because illness in dogs can coincide with
or precede illness in humans.
Etiologic Agent
Rocky Mountain spotted fever (RMSF) is caused by Rickett-
sia rickettsii, an obligately intracellular bacteria in the alpha-
proteobacteria (genus Rickettsia, family Rickettsiaceae, order
Rickettsiales).2 The terminology used to describe rickettsial dis-
ease is confusing and inconsistent due to multiple changes in
the taxonomic classification of organisms in recent years.3 The
300
terms “rickettsial disease,” “rickettsioses,” and even the term
“Rickettsia” have been used to refer to several obligately intra-
cellular organisms including Rickettsia, Bartonella, Ehrlichia,
Anaplasma, Coxiella, and Neorickettsia. Previously, all of these
organisms belonged to the order Rickettsiales and most were
in the family Rickettsiaceae, based on their fastidious or intra-
cellular nature and other characteristics. Therefore, collectively
members of these diverse genera were referred to as “rickett-
sial organisms.”4,5 Recent advances in molecular biologic tech-
niques have resulted in the reclassification of several of these
organisms. Many have been moved out of the family Rickett-
siaceae, and others have been moved out of the order Rickett-
siales. Now the order Rickettsiales includes only two families,
the family Anaplasmataceae, which contains the genera Ana-
plasma, Ehrlichia, Wolbachia, and Neorickettsia; and the fam-
ily Rickettsieaceae, which includes the genera Rickettsia and
Orientia.2,3,5 Currently, “rickettsial” refers to diseases caused
by organisms in the genera Anaplasma, Ehrlichia, Rickettsia,
Neorickettsia, and Orientia, “rickettsioses” refers to diseases
caused by organisms in the family Rickettsiaceae (Rickettsia
and Orientia), and the term “Rickettsia” refers specifically to
members of the genus Rickettsia.3-5
The genus Rickettsia is currently divided into the spotted
fever group (SFG) and the typhus group based on phenotypic
and, more recently, genotypic characteristics.3,5 Some of the
phenotypic characteristics that have historically been used to
group the organisms include the types of vectors that transmit
them and the pathophysiologic manifestations of the disease.3
The SFG rickettsial species are transmitted by arthropod (pri-
marily tick) vectors and infect endothelial cells in mammalian
hosts.5 The two most pathogenic and well-studied SFG rickett-
siae are Rickettsia rickettsii, the cause of RMSF in the Western
Hemisphere, and Rickettsia conorii, the cause of Mediterranean
spotted fever (MSF) in other areas of the world. The first case of
RMSF was described in the late 1800s and the first case of MSF
was described in the 1920s.6 Therefore, these organisms and
their associated diseases are the most well characterized among
the SFG rickettsiae.3 Currently, there are 20 or more species
of SFG rickettsiae.3,6 Some species appear to be nonpathogenic
endosymbionts of ticks.6 However, some SFG rickettsiae previ-
ously thought to be nonpathogenic, such as R. parkerii and R.
massiliae, have recently been associated with disease in people.7-9
Previously thought to have rather limited geographic boundar-
ies, many SFG rickettsiae have also been detected in expand-
ing geographic locales around the world.6 For example, RMSF,
originally described in the Bitterroot Valley of Montana, was
subsequently found to be a frequent tick-transmitted infection
in the eastern United States, but only recently has transmission
via the brown dog tick been documented in the southwestern
 CHAPTER 30  Rocky Mountain Spotted Fever 301

United States.10,11 Increasing travel and the effects of climate

change on tick populations and habitat are thought to be in part
responsible for this phenomenon.12,13

Epidemiology
Hosts, Life Cycle, and Transmission
Pacific coast tick
Dogs are sentinels for SFG rickettsioses in people. Both (Dermacentor occidentalis)

R. rickettsii and R. conorii infect and cause disease in dogs.14-20

Rickettsia rickettsii infection in dogs can occur before, or coin-
American dog tick
cide with, outbreaks of RMSF in people in the same household (Dermacentor variabilis)
or community.21-24 Several serosurveys in endemic areas have
shown an increased risk of MSF in people who live near dogs
that are seropositive for SFG rickettsiae.25-27 Other SFG rickett-
sial species likely infect dogs, but the extent to which they cause
clinical disease has not yet been established. Species implicated
in natural infection in dogs include R. massiliae, R. japonica,
and R. australis.28-31
Young and purebred dogs are overrepresented in some but
not other studies.17,18 Although some studies suggest male dogs
are at increased risk, no sex predilection has been definitively
documented.17,18,32 Severe disease has been reported in English
springer spaniels with phosphofructokinase (PFK) deficiency
and German shepherd dogs.18,32 Although antibodies to SFG
rickettsiae can be detected in cats that live in endemic areas, A
the ability of SFG rickettsiae to actively infect and cause disease
in cats has not been well characterized.33 Similarly, the abil-
ity of typhus group Rickettsia to cause disease both in dogs
and cats has also not been well characterized and will not be
discussed here.
Rickettsia rickettsii is transmitted by several hard (ixodid)
ticks including Dermacentor variabilis (the American dog tick)
(Figure 30-1, A), Dermacentor andersoni (the Rocky Mountain
Rocky Mountain wood tick
wood tick) (Figure 30-1, B), Amblyomma americanum, Ambly- (Dermacentor andersoni)
omma cajennense, Amblyomma aureolatum, and Rhipicephalus
sanguineus.34-38 Amblyomma cajennense and to a lesser extent,
A. aureolatum transmit the rickettsia in South America. Ticks
that transmit R. rickettsii feed once during each life stage (Figure
30-2). Both transstadial and transovarial transmission occurs in
Dermacentor spp. infected with R. rickettsii, so these ticks serve
as a reservoir of infection.39,40 Dermacentor spp. and Ambly-
omma spp. are three-host ticks. Infection is transmitted among
mammalian hosts and ticks when the tick feeds on different hosts
during each stage of molting. The sylvatic cycle for Dermacentor
ticks involves small mammals, such as chipmunks, pine voles,
mice, and ground squirrels. After organisms are ingested by the
tick, SFG rickettsiae initially replicate in the epithelial cells of
B
the tick midgut, enter the hemolymph and hemocytes, and then
FIGURE 30-1  A, Distribution of Dermacentor variabilis and Dermacentor occidenta-
multiply in tissues.40 Once in the salivary glands, the organism
lis ticks in the United States. B, Distribution of Dermacentor andersoni ticks in the United
is transmitted to a naïve mammalian host on feeding. Transmis- States.
sion can occur within hours of attachment. However, this may
be prolonged (up to 48 hours) when changes in virulence occur
under conditions such as starvation of the tick. These “dor- The role of dogs as a natural reservoir of infection for
mant” rickettsiae undergo a process called reactivation after the R. rickettsii is also unknown.39 Dogs are thought to act as inci-
tick begins feeding.41 Therefore, rapid tick removal can decrease dental hosts when Dermacentor ticks are the vectors, because
the risk of transmission in many, but not all, circumstances. ticks rarely acquire the organism from rickettsemic dogs.43 In
Rhipicephalus sanguineus is a one-host tick, with dogs being contrast, all stages of Rh. sanguineus acquired infection at a high
the preferred host.42 Rh. sanguineus can adapt to hot environ- rate from experimentally infected dogs in one study, but this
ments and commonly resides in walls of housing structures in may have been related to the virulence of the rickettsial strain
close proximity to humans.42 It occasionally feeds on humans, used.38 A relatively high infection rate with SFG rickettsiae in
especially when ambient temperatures are high.12 The role of this naturally infected Rh. sanguineus has also been described.37,44
tick as a reservoir for R. rickettsii in nature has yet to be elucidated. Therefore, dogs may play a role in maintenance of a reservoir
302 SECTION 2  Bacterial Diseases

Adults attach to
7
third host for feeding
and mating

Nymphs molt
into adults
6 1 Adult females
drop off host
to lay eggs

5 2 Eggs hatch
Nymphs feed into larvae
on second host
and may acquire
R. rickettsii 3 Larvae feed
4 on first host
Larvae molt and may acquire
into nymphs R. rickettsii
FIGURE 30-2  Transmission cycle of Rickettsia rickettsii in tick populations, people, and dogs. After adult female ticks lay eggs and they hatch (1 and 2), Dermacentor spp. ticks acquire
R. rickettsii upon feeding as larvae or nymphs on rodents (3); they then transmit during subsequent feeding as nymphs or adults (4 and 6). Adult ticks feed on a variety of canid species, such as
foxes and possibly wolves and coyotes. In addition, R. rickettsii infection can be maintained transovarially in Dermacentor ticks, so some larvae that hatch from the egg mass already harbor
an infection. (Redrawn from Nicholson WL, Allen KE, McQuiston JH, et al. The increasing recognition of rickettsial pathogens in dogs and people. Trends Parasitol 2010;26[4]:205-212.)

Alaska

0 cases
1 - 100 cases
Hawaii
100 - 500 cases
500 - 1000 cases
> 1000 cases
FIGURE 30-3  Geographic distribution of the incidence of spotted fever group rickettsioses in people in the United States, 2005-2009. (Compiled from data from the Centers for Disease
Control and Prevention, Morbidity and Mortality Weekly Reports.)

of R. rickettsii infection in endemic foci where Rh. sanguineus
is the primary vector.
Direct transmission of R. rickettsii to people has been
described in laboratory settings through aerosolization. Direct
transmission from blood or other contaminated biologic prod-
ucts also has the potential to occur.
Incidence and Geographic Distribution
Rocky Mountain spotted fever occurs in North, Central, and
South America (where it is known as Brazilian spotted fever).
In North America, most cases of RMSF occur in the southeast-
ern and south central states (Figure 30-3). Disease distribution
in the United States primarily follows the distribution of the
primary vectors, D. variabilis and D. andersoni, although the
importance of D. variabilis as vectors in endemic areas has
recently been questioned.45 Although disease can occur any time
of year, most cases of canine and human RMSF are reported
from April through October, months of peak tick activity.17,46,47
Dogs that live outdoors, particularly those with access to shrubs
and high grass, are at increased risk.17
In recent years the incidence of RMSF in people has
increased.47 Some of the increase may be due to misdiagnosis
of disease caused by species of SFG rickettsiae other than R.
­rickettsii.48,49 A similar increase in seroprevalence rates has been
documented in dogs suspected to have a tick-borne infection in
the United States.50 The geographic distribution of RMSF in
the United States has also increased beyond the distribution
of D. variabilis and D. andersoni. Rhipicephalus sanguineus

is the main vector for R. rickettsii in some parts of the West-
ern Hemisphere, including Mexico and now the south-
western United States. In South America, Amblyomma
species are the main tick vector. Tick species other than
D. variabilis, D. ­andersoni, and Rh. sanguineus are also
infected with R. ­rickettsii in the United States.44 For example,
A. ­americanum transmitted RMSF to a person in North Car-
olina.36 An outbreak in Mexicali, Mexico, was attributed to
R. rickettsii transmitted by Rh. sanguineus ticks associated
with dogs.51 This prompted ongoing surveillance efforts in
ticks, dogs, and people in southern California, a nonendemic
area for RMSF. Thus far, no R. ­rickettsii has been found in
Rh. ­sanguineus ticks from dogs residing in animal shelters
located just across the U.S. border from the outbreak.52,53 This
may be due to subtle differences in microenvironment or other
factors that affect regional infection rates in ticks.54
Clinical Features
Signs and Their Pathogenesis
The endotheliotropism associated with these bacteria results in
the characteristic clinical sign of infection, which is disseminated
vasculitis. In people, cutaneous macules, papules, and petechia-
tion that occur in association with vasculitis form a rash that
looks like “spots,” hence the name Rocky Mountain spotted
fever.6 The clinical signs, time course of illness, and response to
therapy for RMSF in dogs are very similar, if not identical, to
those associated with RMSF in people.17-20,34 However, cutane-
ous lesions are not always present in dogs or people (“Rocky
Mountain spotless fever”). In people, spotted fever rickettsioses
caused by organisms other than R. rickettsii are often associ-
ated with eschar (an area of cutaneous necrosis) formation.2,13
Clinicians should keep in mind that dogs from nonendemic
areas that have classic signs of RMSF, or those that present with
atypical clinical signs, may be infected with R. rickettsii or other
SFG rickettsial species.
Once the organism is inoculated, it spreads through lym-
phatics or directly into the bloodstream to the small capillaries.
R. rickettsii primarily infects endothelial cells, although smooth
muscles and monocytes may also be infected.2,55 The bacterial
outer membrane protein A (OmpA) and outer membrane pro-
tein B (OmpB) are important for attachment, adhesion, and vir-
ulence.56,57 These proteins are also responsible for differences in
serotype, and antibodies to OmpB confer immunity to infection
in experimental settings. SFG rickettsiae enter cells by induc-
ing phagocytosis and are released into the cytoplasm through
the action of enzymes such as phospholipase D and hemolysin
C.57 The bacteria live in the cytoplasm and the nucleus, deriving
nutrients and energy from the host cell.2,56 They spread from
cell to cell by inducing actin to polymerize, which pushes the
bacteria directly into adjacent cells.57,58 This helps them evade
the immune response and to disseminate without rupturing the
cell. They are also released into circulation when they exit the
luminal surface of the cell membrane or when endothelial cell
death or detachment occurs.57 Spotted fever group rickettsiae
activate the transcription factor NFκB, which inhibits apoptosis
and fosters further growth of the organism.56,57
Damage to endothelial cells leads to vasculitis and an increase
in microvascular permeability. Mechanisms of cellular damage
include oxidative injury through the production of reactive
oxygen species, cellular necrosis, and induction of endothelial
apoptosis by CD8+ T cells.55-57,59 Activation of PFK may be
CHAPTER 30  Rocky Mountain Spotted Fever 303
important in maintaining vascular integrity and energy metabo-
lism in endothelial cells under hypoxic or oxidative stress,60,61
which may explain the predisposition of English springer span-
iels with PFK deficiency to severe disease. Vasculitis associated
with R. rickettsii infection manifests as disordered primary
hemostasis, tissue edema, hypovolemia, and microthrombosis.
Increased vascular permeability and the associated edema
and hypovolemia results from disruption of adherens junc-
tions, endothelial cell death, expression of inflammatory cyto-
kines such as Il1-β, IFN-γ, and TNF-α, and induction of COX-2
with subsequent prostaglandin production.55 Microthrombosis
results from altered platelet adherence to endothelium, increased
tissue factor expression, increased plasminogen activator inhibi-
tor, and the release of von Willebrand’s factor.55
Low numbers of organisms circulate in blood for approxi-
mately 13 days after infection, which includes the time that
clinical signs are observed.19,62,63 Organisms are free and also
contained within circulating endothelial cells, which are thought
to be released from the vessels because of decreased adhesion
after rickettsial invasion.57 Thus, RMSF is an acute disease.
Chronic infection has not been documented in dogs or people.
Co-infection with other vector-borne agents is common and
should be considered if the clinical signs are atypical, if there is an
incomplete response to doxycycline therapy, or if clinical signs
have been present for a week before the time of evaluation.17,64
Because of variation in the extent and severity of vascular
injury among dogs, a range of signs can occur, and importantly,
disease manifestations are initially mild and nonspecific.15,17,18
Often, there is no known history of a tick bite. Therefore, the
clinician (physician and veterinarian) must have a high index of
suspicion in order to correctly diagnose and treat this disease.
This is very important because a delay in diagnosis and appropri-
ate antimicrobial therapy dramatically increases morbidity and
mortality in people and in dogs.17,46 A “One Health” approach
to the management of canine and human RMSF is clearly logical.
Lethargy and anorexia are common and may be the only clinical
signs. Vomiting and diarrhea occur frequently in dogs and people
with RMSF. Melena may be observed, as may a variety of CNS
abnormalities including vestibular disease and seizures.17,18,20
Dramatic and rapid weight loss has been described.20
Physical Examination Findings
Fever is present in approximately 80% of naturally infected
dogs. Ocular signs are also frequently observed and may
include a mucopurulent discharge, scleral and conjunctival
injection and hemorrhage, conjunctivitis, uveitis, retinal hem-
orrhage, and retinitis (Figure 30-4). Lymphadenomegaly and
splenomegaly also occur. Respiratory abnormalities include
nasal discharge, epistaxis, and tachypnea. Mucocutaneous and
cutaneous abnormalities include petechiae, ecchymosis, periph-
eral edema (which can be localized over a joint, the prepuce,
or the mammary chain), hyperemia, and necrosis. Gangrenous
necrosis can be so severe as to require reconstructive surgery
after successful treatment of the acute febrile illness.15-18,20,65,66
Orchitis and scrotal edema, hyperemia, and epididymal pain are
common in intact male dogs and should prompt consideration
of RMSF when present. Generalized myalgia and arthralgia can
be observed. Arrhythmias may also be detected. CNS abnor-
malities can be focal or generalized and include paraparesis,
tetraparesis, ataxia, hyperesthesia, ataxia, central or peripheral
vestibular signs, stupor, seizures, and/or coma. Neurologic signs
are more common in dogs with a high R. rickettsii antibody
304 SECTION 2  Bacterial Diseases

titer, which suggests a longer duration of illness or a delay in
diagnosis.17 Residual neurologic deficits may occur after infec-
tion in severely affected individuals. Microvascular hemorrhage,
thrombosis, hypotension, oliguric renal failure, cardiovascular
collapse, and coma can occur terminally.
Diagnosis
A combination of diagnostic testing is often necessary to confirm
infection by SFG rickettsiae. Active infection is confirmed in a
patient with compatible acute clinical signs and demonstration of
the organism using PCR assays or immunohistochemistry (IHC),
or documentation of seroconversion (Figure 30-5). Importantly, a
high index of suspicion based on clinical signs is necessary, because
treatment must be instituted before the results of diagnostic tests
(including rapid PCR assays) confirm infection.17,20 Co-infection
with other vector-borne disease agents should be considered in
patients who fail to respond rapidly to treatment (within the first
24 to 48 hours after initiation of doxycycline treatment).64 Because
direct inoculation into blood or aerosolization can cause infection,
all specimens should be handled with care and marked clearly as
biohazards. Needle-stick injuries, contact with cuts in the skin, and
aerosolization of rickettsemic blood should be avoided.
Laboratory Abnormalities
Complete Blood Count
Thrombocytopenia is common in RMSF and occurs due to vas-
culitis and immune-mediated platelet destruction.67 However,
it does not occur in all dogs with RMSF.17,20 The white blood
cell count may initially decrease and then tends to increase with
duration of illness.18-20 Neutrophils may have toxic change.17
Despite the acute nature of this severe illness, a nonregenera-
tive anemia may be present and persist until the dog is treated
appropriately and begins to recover.15,17,20

Serum Biochemical Tests

Serum biochemical abnormalities can include hypoalbuminemia
(due to vasculitis or protein-losing nephropathy), increased ALP
A activity, hyponatremia, and mild hyperbilirubinemia.15,17,20,65
Hyponatremia has been associated with the syndrome of inap-
propriate antidiuretic hormone secretion (SIADH) in people
with RMSF.

Urinalysis
Urinalysis results in dogs with RMSF are variable and may
include proteinuria, hematuria and bilirubinuria, and pyuria.
Granular casts can be observed.17,18

Coagulation Profile
Coagulation abnormalities include prolonged APTT and
increased serum fibrinogen concentration. Less commonly, pro-
longed PT and increased fibrin degradation products are observed.
Although a prothrombotic state can occur during fulminant dis-
ease, disseminated intravascular coagulation is uncommon.15,17

Body Fluid Cytology

B Cytologic examination of aspirates from enlarged lymph
FIGURE 30-4  Ocular complications in dogs with RMSF. A, Conjunctival hyperemia nodes in dogs with RMSF is consistent with reactive lym-
and scleral injection. B, Retinal hemorrhages. phoid hyperplasia.17,18 Arthrocentesis may reveal neutrophilic

Clinical Signs Convalescence

DPI 0 23 17 36+
Culture

PCR*

Tissue IFA

Serology

Tick bite!
FIGURE 30-5  Timing in relation to day postinfection (DPI) that clinical signs and diagnostic tests for Rickettsia rickettsii may be positive in an infected dog16,62,63,70,76
Further studies are needed to determine exactly how DNA can be detected in peripheral blood after infection.62,63
 CHAPTER 30  Rocky Mountain Spotted Fever 305

polyarthritis.17,18 Cerebrospinal fluid analysis in dogs with neu-
rologic signs may reveal a mixed cellular, neutrophilic, or lym-
phocytic pleocytosis.17,18,20,68
Diagnostic Imaging
Thoracic radiographs in dogs with RMSF may show an unstruc-
tured interstitial pattern.18,69 Testicular ultrasound findings may
be consistent with orchitis in intact males.66
Microbiologic Tests
Diagnostic assays available for RMSF in dogs are shown in
Table 30-1. Because of the pathogenesis of the infection, labo-
ratory diagnostics (including serology and PCR assays) may not
indicate infection is present at the time that clinical signs mani-
fest (see Figure 30-5). In addition, most diagnostic tests do not
differentiate among species of SFG rickettsiae.
Culture
Because of the obligately intracellular and extremely pathogenic
nature of R. rickettsii, culture is difficult to perform and process-
ing of specimens for isolation is limited to BSL 3 facilities (see
Table 1-4). Therefore, culture is not commonly used for routine
diagnosis. Currently, R. rickettsii is listed as a Category A Bio-
terrorism agent, so all isolates must be reported to the Centers
for Disease Control and Prevention or destroyed immediately.
Serologic Diagnosis
Indirect microimmunofluorescence (MIF) assays that detect
IgM and IgG antibodies are most commonly used to document
seroconversion in dogs infected with R. rickettsii.19 Because
of a lack of specificity, assays that detect IgM alone cannot be
used to accurately diagnose acute RMSF.70 There is extensive
serologic cross-reactivity among SFG rickettsiae, and thus
a positive titer only indicates exposure to a SFG rickettsial
species. In general, the infecting species is presumed based
on geographic locale, so RMSF is the presumptive diagnosis
in dogs that seroconvert in the southeastern United States.
However, other species of SFG rickettsiae may also be pres-
ent and induce disease.28,71 Exposure to nonpathogenic SFG
rickettsiae, some of which are common endosymbionts in
ticks, may be a common cause of positive titers, particularly
low and persistent titers, to R. rickettsii in healthy people
and dogs.49,72 Serologic cross-reactivity with SFG rickettsiae
also may occur in dogs and people infected with Bartonella
henselae.73 In addition, previous infection with R. rickettsii
may result in persistent antibody titers.70 Thus, infection
with R. rickettsii cannot be definitively diagnosed based on
a single positive titer. However, a single high titer in the con-
text of acute and compatible clinical signs and an appropriate
response to therapy suggests infection.17 Titers are commonly
negative early in the course of infection, because clinical signs
often occur before seroconversion in naturally and experi-
mentally infected dogs.20,70,74 Thus, acute and convalescent
serology (run in the same laboratory and ideally in the same
batch) and documentation of a fourfold change in titer is nec-
essary to confirm acute SFG rickettsiae infection. The conva-
lescent serum specimen should be drawn 2 to 3 weeks after
the acute specimen.

TABLE 30-1
Diagnostic Assays Available for Rocky Mountain Spotted Fever in Dogs
Assay Specimen Type Target Performance
Indirect Serum IgM and IgG a­ ntibodies Demonstration of a fourfold change in titer (seroconversion)
microimmuno­ against R. rickettsii is very sensitive. False-negative results are common during
fluorescence the acute phase of infection. A positive antibody response
assay (MIF) may reflect prior exposure to a nonpathogenic SFG rick-
ettsia, or long-lived circulating antibody from previous
infection with SFG rickettsiae. Cross-reactivity to other
SFG rickettsiae and to B. henselae occurs.
PCR Whole blood Gene target varies In vitro sensitivity may approach 100%; however, absolute
with testing (clinical) sensitivity is lower, approximately 60%, because
laboratories organisms circulate in blood in low numbers and tran-
siently during the acute phase of infection. Sensitivity is fur-
ther reduced by antimicrobial drug treatment. Specificity
approaches 100%, particularly if the laboratory sequences
all PCR amplicons. PCR testing for other tick-borne
organisms that cause similar clinical signs is available as
PCR panels from some testing laboratories. Only a few
PCR assays differentiate among Rickettsia species. Nega-
tive PCR assay results do not rule out infection.
Histopathology Cutaneous biopsy Characteristic Direct IFA sensitivity is approximately 80% during acute
with direct IFA (inguinal or flank ­perivascular infection. Biopsy of a petechial or ecchymotic lesion may
or Gimenez targeting areas ­inflammation and increase sensitivity. False negatives occur if organisms are
­staining with lesions) ­vasculitis with absent from the lesion because of random chance, timing
or necropsy ­necrosis and p
­ resence of specimen collection, or prior antimicrobial treatment.
­specimens of the organism Does not differentiate among species of SFG Rickettsia.
306 SECTION 2  Bacterial Diseases
Molecular Diagnosis Using Polymerase Chain Reaction
PCR testing of whole blood can be used to confirm infection
in seronegative dogs during the acute phase of disease.62,63,74
A sensitive PCR assay that detects and differentiates among
SFG rickettsiae that infect dog blood has been described, and a
variety of PCR assays are available through commercial veteri-
nary diagnostic laboratories.74 However, because R. ­rickettsii
circulates in blood in low numbers and only transiently, the
sensitivity of any diagnostic assay to detect infection in the
bloodstream is limited. One real-time PCR assay, which could
detect as few as five copies of organism DNA, had a sensitiv-
ity of only 53% for detection of R. rickettsii in acute samples
from naturally infected dogs with signs of RMSF.74 The sen-
sitivity of another R. rickettsii PCR assay for detection of
R. rickettsii in experimentally infected dogs ranged from 33% to
100% depending on the day of sampling.63 Furthermore, false
negatives can occur after initiation of appropriate antimicrobial
treatment. Therefore, although a positive PCR assay result con-
firms infection, a negative result does not exclude the diagnosis.
Some PCR assays can differentiate among infecting Rickettsia
species, whereas others only amplify conserved rickettsial DNA
targets.62,74 Clinicians should consult with laboratories to deter-
mine the in  vitro (analytical) and in  vivo (clinical) sensitivity
of a PCR assay and its ability to differentiate among infecting
Rickettsia species.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with RMSF are consistent
with vasculitis and may include edema, particularly of the
ears, muzzle, and scrotum, and ecchymosis, petechiae, and/or
focal hemorrhages of the skin, mucous membranes, and viscera
(including the brain). Lymphadenomegaly and splenomegaly
are also common findings.15,16,20,22
Histopathologic Findings
Microscopically, the predominant lesion is vasculitis. Neutro-
phils, monocytes, and/or lymphoreticular cells predominate in
the inflammatory lesions. The inflammation may surround and
invade large small and medium-size vessels of multiple organs
and tissues and is frequently accompanied by focal necrosis
and hemorrhage. Meningoencephalitis, splenitis, myocardial
necrosis, glomerulonephritis, and renal vasculitis have been
described.15,20,22
Direct IFA can be used to detect organisms in skin and other
organs. Identification of abundant SFG rickettsiae within and
around small to medium-size vessels and vascular endothelial
cells can confirm infection.20 In experimentally infected dogs,
the sensitivity of IHC was 78.3%, and organisms could be
visualized between days 7 and 12 of infection (days 3 to 8 of
fever).75 In that study, specimens collected from areas other
than the central focus of vasculitis were negative. However, in
naturally infected dogs, the sensitivity was 80% for skin speci-
mens from the inguinal region. Half of those specimens lacked
gross lesions.18 Thus, the diagnostic sensitivity of IHC is most
likely enhanced by obtaining lesional biopsies, but may not be
decreased when a lesion is not biopsied. The sensitivity of direct
IHC is decreased by antibiotic therapy. IHC does not differenti-
ate between SFG rickettsial species. Gimenez stain can also be
used to visualize rickettsial organisms in tissues or tissue culture
isolation attempts, but this stain is not rickettsia specific and
does not allow for differentiation among species.
Treatment
Antimicrobial Treatment
Appropriate antibiotic therapy must be immediately instituted
based on clinical suspicion, and before diagnostic tests con-
firm infection. Inappropriate or delayed antibiotic therapy may
increase morbidity and mortality.17,46 Some antimicrobial drugs
such as trimethoprim sulfonamides may actually worsen dis-
ease progression in human patients.46 Doxycycline is the treat-
ment of choice (Table 30-2). It effectively eliminates infection
and is active against A. phagocytophilum, Ehrlichia spp., and
B. ­burgdorferi, which may be present in co-infections or cause
disease that resembles RMSF. Seven days of treatment is ade-
quate in most cases. Treatment a few days past defervescence is
recommended. A longer course of treatment is recommended for
dogs that are co-infected with Ehrlichia spp. or B. ­burgdorferi.
Chloramphenicol was effective in experimentally infected
dogs.76 However, it may be less effective than doxycycline for
treating RMSF in people and has less activity in  vitro against
Ehrlichia chaffeensis and A. phagocytophilum.46 Enrofloxacin
was effective for treatment of RMSF in experimentally infected
dogs.76 However, enrofloxacin is not effective for treatment of
E. canis infections.77 Use of parenteral antimicrobial drugs may
be necessary in severely debilitated or vomiting patients.
Supportive Care
Many dogs require hospitalization.20 Aggressive supportive care
for complications such as thrombosis, CNS deficits, and gastro-
intestinal signs may be necessary. Because of the loss of vascular
integrity, fluids should be administered with caution, and colloids
may be warranted in some cases. The clinician should avoid exac-
erbation of interstitial edema, which can contribute to cerebral
edema and death. The use of glucocorticoids in dogs with RMSF
is controversial. Antiinflammatory and immunosuppressive doses
did not affect overall outcome experimentally infected dogs, but
rickettsemia was prolonged in dogs concurrently treated with
immunosuppressive doses of glucocorticoids and doxycycline.78
Antiinflammatory doses of glucocorticoids have been used in
dogs with severe CNS manifestations and may be necessary topi-
cally or systemically for treatment of ocular abnormalities.68,79
Prognosis
The response to appropriate antibiotic therapy in dogs
with RMSF is rapid (24 to 48 hours). Co-infection with
TABLE 30-2
Antimicrobials Used to Treat Rocky Mountain Spotted
Fever in Dogs
Dose Interval Duration
Drug (mg/kg) Route (hours) (days)
Doxycycline 5 PO, IV 12 7 to 14
Chloramphenicol* 30 PO, IV 8 7 to 14
Enrofloxacin 3† to 5 PO, IV 12 7 to 14
*May cause aplastic anemia in humans (wear gloves); may be less
­effective than doxycycline.
†Dose used in experimentally infected dogs.76 Caution required in young
animals (see Chapter 8).

B. burgdorferi, Ehrlichia spp., B
­ abesia spp., and Bartonella spp.
should be considered in dogs with severe or prolonged clinical
signs or dogs that fail to respond to doxycycline. Residual CNS
and other deficits may occur in severely affected patients.17 The
prognosis is good to excellent if the disease is diagnosed and
treated with appropriate antibiotics and supportive care early in
the course of illness.17,68
Immunity and Vaccination
Cell-mediated and innate immunity is important in the clearance
of rickettsial infections. CD4+ and CD8+ cells, along with macro-
phages and dendritic cells, are believed to be sources of inflamma-
tory cytokines such as IFN-γ, TNF-α, Il-1β, and RANTES (CCL5).
These cytokines increase production of nitric oxide synthetase and
hydrogen peroxide by the endothelial cell, which helps to elimi-
nate the organisms.56,57 The endothelial cells themselves also pro-
duce cytokines such as IL-6, Il-8, and MCP-1 (CCL2) that recruit
immune cells and combat infection. Antibodies are not thought
to be important initially for clearance of infection because they
form after the fulminant stages of disease.57 However, antibod-
ies are long-lived and together with cell-mediated immunity may
prevent subsequent infection. Immunity to reinfection is thought
to be lifelong in people, and experimentally infected dogs did not
develop illness following rechallenge at 6 months and 3 years
after the initial infection. Vaccination is not available for dogs or
people currently, but may be in the future.80
Prevention
Avoidance of tick-infested areas and routine inspection of dogs
for ticks after outdoor activities can help to prevent RMSF. The
reader is referred to Chapter 28 for information on tick preven-
tion and safe tick removal. Safe tick removal is especially critical
CASE EXAMPLES
Rocky Mountain Spotted Fever in a Dog
Signalment: A 5-year-old MC Australian shepherd from North
Carolina
History: The dog was examined by the North Carolina State
University veterinary emergency service on April 11 because
of recent onset of lethargy, fever, tachypnea, and a stiff gait
when walking. Historically the dog had been healthy, had
received routine vaccinations, heartworm preventive, and a
flea acaricide, and ran approximately 3 miles each evening
with the owner, who was a veterinarian. On the day of
presentation and several hours after the evening run, the
dog became lethargic, refused to eat dog food or treats, and
seemed painful upon manipulation. The owner noticed that
the dog’s heart rate, which was normally 50 to 60 beats/min,
was 120 beats/min at rest. Rectal temperature at home just
prior to presentation was 104.4°F (40.2°C). Three days before
the dog was evaluated, the owner had removed a non-
engorged tick from the dog.
Physical Examination: Physical examination findings
included a body weight of 21.3 kg, 3%-5% dehydration,
CHAPTER 30  Rocky Mountain Spotted Fever 307
when exposure to R. rickettsii is a possibility. In cases where
Rh. sanguineus is the vector, environmental control of ticks is
particularly important.
Public Health Aspects
People living near dogs and with dogs in endemic areas are
at increased risk for acquiring RMSF. This is likely due to
increased human contact with ticks through interaction with
tick-infested dogs. Also, because of transovarial transmis-
sion, dogs and people can independently acquire the infec-
tion from different ticks questing in the same environment.
Because dogs have higher exposure to ticks than their human
counterparts, they serve as excellent environmental sentinels
for RMSF.21,22,24,71 In the United States, the diagnosis of SFG
rickettsioses in humans is notifiable to public health authori-
ties. Some counties in certain states also require reporting
for dogs, particularly during suspected outbreaks.81 From a
public health standpoint, it is important for veterinarians to
confirm a diagnosis of RMSF in a dog whenever possible, and
to warn the family of the increased risk of acquiring a R. rick-
ettsii–infected tick in the same location as the dog acquired
the disease. Veterinarians must communicate with owners and
physicians that infection in a dog may precede tick-transmitted
infection to owners or neighbors. Clients should be instructed
to remove ticks properly. Education of owners with regard to
the importance of tick control and prevention for both peo-
ple and pets and the environment is critical. Novel and well-
characterized species of SFG rickettsiae are important causes
of emerging infectious disease in humans. Furthermore, R.
rickettsii is considered a potential bioterrorist agent.80 Because
dogs are sentinels for infection, veterinarians play an impor-
tant role in detecting, defining, and preventing illness in their
canine patients and their human companions.
pain score of 1 out of 4, rectal temperature of 103.7°F
(39.8°C), HR 130 beats/min, tachypnea (42 breaths/min),
and a normal CRT. The body condition score was 5/9.
The dog stood in a hunched position and had a stiff gait,
but localizing pain was not elicited on palpation of the
spine, joints, or long bones. There was no nasal or ocular
discharge, no petechiae or rash, and no obvious swelling
or joint effusion. Popliteal lymph nodes were prominent,
but there were no other abnormalities noted on physical
examination. Systolic blood pressure was 153 mm Hg, and
diastolic and mean blood pressures were 93 mm Hg and
111 mm Hg, respectively.
Laboratory Findings: The hematocrit was 42%, total
plasma protein was 6.2 mg/dL, and the platelet count was
120,000/µL. A serum biochemical profile showed only
hypoglobulinemia (1.7 g/dL).
Microbiologic Testing: A SNAP 4Dx (IDEXX Laboratories)
in-house ELISA assay was negative. Indirect IFA assays for
Babesia canis, Bartonella henselae, Bartonella vinsonii subsp.
berkhoffii, and Ehrlichia canis were negative (no detectable
antibodies at a 1:16 screening dilution). The dog was
seroreactive to Rickettsia rickettsii antigens at a titer of 1:64.
A PCR panel capable of detecting Anaplasma spp., Babesia
308 SECTION 2  Bacterial Diseases
spp., Ehrlichia spp., and Rickettsia spp. was negative on
whole blood. A convalescent R. rickettsii antibody titer was
1:256, which supported a diagnosis of SFG rickettsiosis,
most likely RMSF.
Treatment: Because of the acute onset of lethargy and fever,
the history of recent tick attachment, the documentation
of thrombocytopenia, and the relatively high frequency of
RMSF in dogs and people in North Carolina, doxycycline
(4.8 mg/kg PO q12h) was prescribed for 3 weeks before
the results of microbiologic testing became available. The
dog responded rapidly after doxycycline treatment, with
appetite and behaviors normalizing within 24 to 48 hours
after the first dose.
Comment: Although the clinical signs in this dog were
relatively nonspecific, the clinical presentation was very
typical of RMSF. Because R. rickettsii causes generalized
vascular injury resulting in increased vascular permeability,
fluid and protein leakage out of the intravascular space, and
an acute neutrophilic inflammatory response throughout
tissue sites within the body, pain that appears to shift
in location (back pain, joint pain, abdominal pain, neck
pain) is a typical disease manifestation. Also, the hunched
appearance reported in this dog is commonly observed
in dogs with RMSF. As this dog was owned by veterinarian
and a tick had been removed a few days earlier, a clinical
diagnosis and appropriate treatment were initiated earlier in
the course of illness than is typical of most dogs with RMSF.
The rapid initiation of doxycycline may have blunted the
humoral antibody response and decreased the convalescent
antibody titer to a level that is lower than expected in most
cases of RMSF in dogs. It is important to note that PCR assay
is insensitive in the early stages of RMSF, as there are often
inadequate numbers of organisms in the blood to achieve
successful amplification of rickettsial DNA. A very rapid
response to doxycycline is expected, unless there has been a
delay in diagnosis, accompanied by the onset of neurologic
abnormalities. Dogs that develop neurologic complications
experience a more prolonged recovery and can have
residual neurologic deficits.
Rocky Mountain Spotted Fever in a Human Patient
On May 9, a 61-year-old man, who resided on a farm in Wake
County, North Carolina, removed an embedded tick from
the hairy portion of his right armpit. Although duration of
attachment was unknown, it was likely that the tick was
acquired 2 to 3 days earlier while pulling weeds from a hay
field. Using a tick identification key, an experienced research
technician classified the tick as a male ­Amblyomma ameri-
canum and stored the tick in a vial containing alcohol. On
May 16, while working on the farm, the patient experienced
mild nausea after drinking water and became transiently
dizzy. The next morning chills developed, and by the mid-
afternoon the patient became febrile (101.2°F [38.4°C]),
developed muscle pain, a mild headache and remained in
bed until the next morning. These symptoms became pro-
gressively more severe during the day and the tick attach-
ment site had developed into an erythematous, circular le-
sion with induration and a necrotic center, consistent with a
rickettsial eschar. Due to the history of tick attachment and
fever (maximum temperature 102.9°F [39.4°C]), a spotted
fever rickettsiosis, such as RMSF, or neutrophilic or mono-
cytic ehrlichiosis, caused by Ehrlichia ewingii and Ehrlichia
chaffeensis, respectively, was suspected. Neutropenia (2553
cells/µL) was accompanied by a left shift (5% band neutro-
phils) and thrombocytopenia (148,000/µL). Doxycycline
was dispensed with instructions to take 100 mg q12h for
7 days. That evening, after sleeping for 6 hours, the patient
ate a small quantity of food, after which he became severely
nauseated and fainted, and impact with the floor was asso-
ciated with a severe blow to the back of the head. The next
day (May 19) a maculopapular rash appeared that predomi-
nantly involved the inferior portions of the arms and legs.
Throughout the day the distribution of the rash spread and
the severity progressed from barely visible to obvious over
most of the body. At no time did the rash involve the palms
or plantar surface of the feet. Fever resolved within 36 hours
after the initiation of doxycycline, and the rash began to
fade gradually after 48 hours of treatment. Within a week
after starting doxycycline, the patient was experiencing
no symptoms and had no sequelae as a result of the infec-
tion or the fall. A repeat CBC (May 25) identified a normal
neutrophil count (4378 cells/µL), no band neutrophils, and
a normal platelet count (320,000/µL). Acute-phase serum
was collected and stored until the convalescent sample was
obtained. Rickettsia and Ehrlichia spp. PCR was performed
immediately. PCR amplicons, obtained from the patient’s
blood and from the tick, were sequenced, confirming the
presence of R. rickettsii DNA. PCR for Ehrlichia spp. DNA was
negative from both the tick and the patient. Subsequently,
seroconversion to R. rickettsii antigens was identified (1:64
acute titer, convalescent titer 1:512 after 4 weeks). The pa-
tient did not seroconvert to Ehrlichia spp. antigens.
Comment: Historically, transmission of R. rickettsii in North
America was attributed solely to Dermacentor variabilis in
the eastern United States and to Dermacentor andersoni
in the western United States. However, between 2002 and
2004, researchers at the Centers for Disease Control and
Prevention documented Rh. sanguineus transmission of R.
rickettsii to people residing in Arizona. Although this is most
likely an infrequent occurrence, this patient was infected
with R. rickettsii by A. americanum. It is important to note
the similarities between the historical, physical examination
and laboratory findings for the dog discussed previously
and the human patient. In several published case reports,
dogs develop RMSF before a family member contracts the
infection from a tick. Thus, it is important for veterinarians
to recognize and confirm a diagnosis of RMSF in dogs and to
educate the client as to the risk of tick exposure within their
local environment.

SUGGESTED READINGS
Gasser AM, Birkenheuer AJ, Breitschwerdt EB. Canine Rocky Moun-
tain spotted fever: a retrospective study of 30 cases. J Am Anim Hosp
Assoc. 2001;37(1):41-48.
Elchos BN, Goddard J. Implications of presumptive fatal Rocky Moun-
tain spotted fever in two dogs and their owner. J Am Vet Med Assoc.
2003;223(10):1450-1452:1433.
Nicholson WL, Allen KE, McQuiston JH, et al. The increasing recogni-
tion of rickettsial pathogens in dogs and people. Trends Parasitol.
2010;26(4):205-212.
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23. Demma LJ, Traeger M, Blau D, et al. Serologic evidence for expo-
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24. Nicholson WL, Gordon R, Demma LJ. Spotted fever group rickett-
sial infection in dogs from eastern Arizona: how long has it been
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25. Raoult D, Toga B, Dunan S, et  al. Mediterranean spotted fever
in the South of France; serosurvey of dogs. Trop Geogr Med.
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26. Mannelli A, Mandola ML, Pedri P, et  al. Associations between
dogs that were serologically positive for Rickettsia conorii relative
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27. Segura-Porta F, Diestre-Ortin G, Ortuno-Romero A, et  al. Prev-
alence of antibodies to spotted fever group rickettsiae in human
beings and dogs from an endemic area of Mediterranean spotted
fever in Catalonia, Spain. Eur J Epidemiol. 1998;14(4):395-398.
28. Beeler E, Abramowicz KF, Zambrano ML, et al. A focus of dogs
and Rickettsia massiliae–infected Rhipicephalus sanguineus in Cal-
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29. Sexton DJ, Banks J, Graves S, et  al. Prevalence of antibodies to
spotted fever group rickettsiae in dogs from southeastern Australia.
Am J Trop Med Hyg. 1991;45(2):243-248.
30. Satoh H, Tsuneki A, Inokuma H, et al. Seroprevalence of antibod-
ies against spotted fever group Rickettsia among dogs and humans
in Okinawa, Japan. Microbiol Immunol. 2001;45(1):85-87.
31. Inokuma H, Matsuda H, Sakamoto L, et  al. Evaluation of Rick-
ettsia japonica pathogenesis and reservoir potential in dogs by
experimental inoculation and epidemiologic survey. Clin Vaccine
Immunol. 2011;18(1):161-166.
32. Greene CE, Breitschwerdt EB. Rocky Mountain spotted fever,
murine typhus-like disease, rickettsialpox, typhus, and Q fever. In:
Greene CE, ed. Infectious Diseases of the Dog and Cat. 6th ed.
Philadelphia, PA: Saunders Elsevier; 2006:232-241.
33. Bayliss DB, Morris AK, Horta MC, et al. Prevalence of Rickettsia
species antibodies and Rickettsia species DNA in the blood of cats
with and without fever. J Feline Med Surg. 2009;11(4):266-270.
34. Warner RD, Marsh WW. Rocky Mountain spotted fever. J Am Vet
Med Assoc. 2002;221(10):1413-1417.
35. Guedes E, Leite RC, Prata MC, et al. Detection of Rickettsia rick-
ettsii in the tick Amblyomma cajennense in a new Brazilian spotted
fever-endemic area in the state of Minas Gerais. Mem Inst Oswaldo
Cruz. 2005;100(8):841-845.
36. Breitschwerdt EB, Hegarty BC, Maggi RG, Lantos PM, Aslett DM,
Bradley JM. Rickettsia rickettsii Transmission by a Lone Star Tick,
North Carolina. Emerg Infect Dis. 2011;17(5):873-875.
37. Demma LJ, Traeger MS, Nicholson WL, et  al. Rocky Mountain
spotted fever from an unexpected tick vector in Arizona. N Engl J
Med. 2005;353(6):587-594.
38. Piranda EM, Faccini JL, Pinter A, et al. Experimental infection of
Rhipicephalus sanguineus ticks with the bacterium Rickettsia rick-
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Dis. 2011;11(1):29-36.
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39. Chen LF, Sexton DJ. What’s new in Rocky Mountain spotted
fever? Infect Dis Clin North Am. 2008;22(3):415-432:vii-viii.
40. Dantas-Torres F. Rocky Mountain spotted fever. Lancet Infect Dis.
2007;7(11):724-732.
41. Kidd LB. Transmission times and prevention of tick-borne diseases
in dogs. Compend Contin Educ Pract Vet. 2003;25(10):742-751.
42. Dantas-Torres F. Biology and ecology of the brown dog tick. Rhipi-
cephalus sanguineus. Parasit Vectors. 2010;3:26.
43. Norment BR, Burgdorfer W. Susceptibility and reservoir poten-
tial of the dog to spotted fever-group rickettsiae. Am J Vet Res.
1984;45(9):1706-1710.
44. Wikswo ME, Hu R, Metzger ME, Eremeeva ME. Detection of Rick-
ettsia rickettsii and Bartonella henselae in Rhipicephalus sanguin-
eus ticks from California. J Med Entomol. 2007;44(1):158-162.
45. Stromdahl EY, Jiang J, Vince M, Richards AL. Infrequency of Rick-
ettsia rickettsii in Dermacentor variabilis removed from humans,
with comments on the role of other human-biting ticks associated
with spotted fever group Rickettsiae in the United States. Vector
Borne Zoonotic Dis. 2011;11(7):969-977.
46. Chapman AS, Bakken JS, Folk SM, et al. Diagnosis and manage-
ment of tickborne rickettsial diseases: Rocky Mountain spotted
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guide for physicians and other health-care and public health pro-
fessionals. MMWR Recomm Rep. 2006;55(RR-4):1-27.
47. Centers for Disease Control and Prevention. Rocky Mountain
Spotted Fever, Statistics and Epidemiology. http://www.cdc.gov
/rmsf/stats:Last accessed May 27, 2012.
48. Moncayo AC, Cohen SB, Fritzen CM, et  al. Absence of
­Rickettsia rickettsii and occurrence of other spotted fever
group rickettsiae in ticks from Tennessee. Am J Trop Med Hyg.
2010;83(3):653-657.
49. Raoult D, Parola P. Rocky Mountain spotted fever in the USA: a
benign disease or a common diagnostic error? Lancet Infect Dis.
2008;8(10):587-589.
50. Diniz PPVP, unpublished data. 2012.
51. Eremeeva ME, Zambrano ML, Anaya L, et  al. Rickettsia rick-
ettsii in Rhipicephalus ticks, Mexicali. Mexico. J Med Entomol.
2011;48(2):418-421.
52. Tinoco-Gracia L, Quiroz-Romero H, Quintero-Martinez MT,
et  al. Prevalence of Rhipicephalus sanguineus ticks on dogs in a
region on the Mexico-USA border. Vet Rec. 2009;164(2):59-61.
53. Fritz CL, Kriner P, Garcia D, et  al. Tick infestation and spotted
fever group Rickettsia in shelter dogs, California, 2009. Zoonoses
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54. Telford SR. Status of the “East Side hypothesis” (transovarial inter-
ference) 25 years later. Ann N Y Acad Sci. 2009;1166:144-150.
55. Sahni SK, Rydkina E. Host-cell interactions with pathogenic Rick-
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56. Walker DH, Valbuena GA, Olano JP. Pathogenic mechanisms of
diseases caused by Rickettsia. Ann N Y Acad Sci. 2003;990:1-11.
57. Walker DH. Rickettsiae and rickettsial infections: the current state
of knowledge. Clin Infect Dis. 2007;45(suppl 1):S39-S44.
58. Balraj P, Renesto P, Raoult D. Advances in rickettsia pathogenicity.
Ann N Y Acad Sci. 2009;1166:94-105.
59. Eremeeva ME, Liang Z, Paddock C, et al. Rickettsia rickettsii infec-
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quantitation of antioxidant enzyme gene expression by RT-PCR.
Ann N Y Acad Sci. 2003;990:468-473.
60. Cummiskey JM, Simon LM, Theodore J, Ryan US, Robin ED.
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61. Asahina T, Kashiwagi A, Nishio Y, et  al. Impaired activation
of glucose oxidation and NADPH supply in human endothe-
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1995;44(5):520-526.
62. Breitschwerdt EB, Papich MG, Hegarty BC, et al. Efficacy of dox-
ycycline, azithromycin, or trovafloxacin for treatment of experi-
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Chemother. 1999;43(4):813-821.
63. Piranda EM, Faccini JL, Pinter A, et  al. Experimental infec-
tion of dogs with a Brazilian strain of Rickettsia rickettsii:
clinical and laboratory findings. Mem Inst Oswaldo Cruz.
2008;103(7):696-701.
64. Kordick SK, Breitschwerdt EB, Hegarty BC, et al. Coinfection with
multiple tick-borne pathogens in a Walker Hound kennel in North
Carolina. J Clin Microbiol. 1999;37(8):2631-2638.
65. Greene CE. Infectious Diseases of the Dog and Cat. 3rd ed. St.
Louis, MO: Saunders Elsevier; 2006.
66. Ober CP, Spaulding K, Breitschwerdt EB, et  al. Orchitis in two
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2004;45(5):458-465.
67. Grindem CB, Breitschwerdt EB, Perkins PC, et  al. Platelet-asso-
ciated immunoglobulin (antiplatelet antibody) in canine Rocky
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1999;35(1):56-61.
68. Mikszewski JS, Vite CH. Central nervous system dysfunction asso-
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J Am Anim Hosp Assoc. 2005;41(4):259-266.
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radiographic findings in dogs infected with Rickettsia rickettsii. Vet
Radiol Ultrasound. 1997;38(4):260-266.
70. Breitschwerdt EB, Levy MG, Davidson MG, et al. Kinetics of IgM
and IgG responses to experimental and naturally acquired Rickettsia
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72. Breitschwerdt EB, Moncol DJ, Corbett WT, et  al. Antibodies to
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73. Solano-Gallego L, Bradley J, Hegarty B, et al. Bartonella henselae
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74. Kidd L, Maggi R, Diniz PP, et  al. Evaluation of conventional
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76. Breitschwerdt EB, Davidson MG, Aucoin DP, et  al. Efficacy of
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77. Neer TM, Eddlestone SM, Gaunt SD, Corstvet RE. Efficacy of
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78. Breitschwerdt EB, Davidson MG, Hegarty BC, et al. Prednisolone
at anti-inflammatory or immunosuppressive dosages in conjunc-
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2009;3(2):4.
CHAPTER 31

Salmon Poisoning Disease

Jane E. Sykes

the distribution of an aquatic snail, Oxytrema silicula (also known

Overview of Salmon Poisoning Disease
First Described: Northwestern Oregon, United States, 1814
(Astoria)1
Cause: Neorickettsia helminthoeca, a gram-negative, obli-
gately intracellular bacteria that belongs to the family
Anaplasmataceae
Affected Hosts: Dogs, foxes, coyotes, raccoons, captive bears
Intermediate Hosts: Aquatic snails, fish (especially salmonids)
Geographic Distribution: Coastal areas of Washington, ­Oregon,
northern California, British Columbia, Brazil (2004)
Route of Transmission: Ingestion of the infected trematode
vector Nanophyetus salmincola, most often in salmonid
fish
Major Clinical Signs: Fever, lethargy, anorexia, vomiting, diar-
rhea, lymphadenomegaly
Differential Diagnoses: Lymphoma, canine parvovirus infec-
tion, canine distemper virus infection, granulocytic
anaplasmosis, ehrlichiosis, leptospirosis, septic shock,
disseminated fungal disease (especially cryptococco-
sis), hemorrhagic gastroenteritis, dietary indiscretion,
gastrointestinal foreign body, pancreatitis, hypoadreno-
corticism, inflammatory bowel disease. Appropriate anti-
microbial treatment with doxycycline must begin before
the diagnosis is confirmed by laboratory testing. Misdi-
agnosis and delayed or inappropriate antimicrobial drug
therapy increase morbidity and mortality.
Human Health Significance: Nanophyetus salmincola can
cause gastrointestinal disturbances and eosinophilia
in humans, but N. helminthoeca does not cause human
disease.
as Juga silicula), which is an intermediate host of the trematode
(Figure 31-1). The snail is prevalent in coastal streams. A similar
disease has been described in dogs from Brazil.6,7 Two related
yet antigenically distinct organisms have been isolated from
dogs with SPD, N. helminthoeca and the Elokomin fluke fever
(EFF) agent.8-12 The EFF agent may be a less pathogenic strain of
N. helminthoeca.
Dogs usually become infected with N. helminthoeca when
they ingest encysted trematode metacercariae within uncooked
or undercooked freshwater fish, most commonly salmonid fish.
The Pacific giant salamander is also a competent second inter-
mediate host. Ingestion of encysted metacercariae is followed
by maturation of the trematode, which feeds on the intestinal
mucosa and inoculates the rickettsia into the host. The fluke
has an oral sucker and a ventral sucker, which are used to grasp
host intestinal tissue, although the fluke itself does not cause
extensive damage to the intestinal wall.13 Infected trematode
ova are shed in the stool for 60 to 250 days.14 After several
months, miracidia develop within the eggs, hatch, swim away,
and penetrate the snail. They then develop into rediae, each
of which give rise to many free-swimming cercariae. Thou-
sands of cercariae are released intermittently from the snail.
These rapidly penetrate the skin of a fish (or are ingested by
the fish), and encyst throughout the fish as metacercariae

Etiologic Agent and Epidemiology

Salmon poisoning disease (SPD) is caused by the rickettsial
pathogen Neorickettsia helminthoeca, which, like other Neor-
ickettsia spp., resides within a trematode (fluke) vector for the
course of the trematode’s life cycle. The trematode vector of
N. helminthoeca is Nanophyetus salmincola, thought to be the
most common trematode species endemic to the United States.2
The disease occurs in dogs, foxes, and coyotes and has also been
reported in captive bears, but does not occur in domestic cats.3-5
SPD is geographically restricted to coastal regions of northern FIGURE 31-1  Geographic distribution of salmon poisoning disease in dogs from
California, Oregon, and Washington in the United States, and the United States. The distribution also extends into the southern portion of ­British
southern British Columbia in Canada (Figure 31-1). This reflects Columbia.

311
312 SECTION 2  Bacterial Diseases

Adult fluke within

intestinal tract
feeds on lining
and injects
bacteria
Eggs
in feces
Metacercaria
encysted in
salmonid fish
A
Miracidium

Snail

Cercaria swims to and

penetrates salmonid fish
FIGURE 31-2  Life cycle of Nanophyetus salmincola, the trematode vector of salmon
poisoning disease. The fluke harbors the rickettsial organism throughout its lifecycle.
(Figures 31-2 and 31-3). Infected fish may contain more than
1000 metacercariae. Infected fish may have visible damage to
their skin, and the infection may also interfere with their swim-
ming activity.15 Dog-to-dog transmission of N. helminthoeca
has been demonstrated experimentally by mechanical aerosol-
ization or rectal administration of lymph node suspensions or
homogenates of rectal mucosa.16
Intact male dogs and Labrador retrievers appear to be over-
represented among dogs with SPD,17 possibly because they are
popular with individuals engaged in fishing activities, although
dogs of any sex and breed can be affected. Dogs of any age
are affected; the median age in one study was 3 years.17 Dogs
develop SPD at any time of year. Occasionally dogs from areas
not endemic for SPD develop disease after they ingest fish that is
imported from endemic areas.18
Most but not all infected dogs have a history of access to
or consumption of raw or improperly cooked fish.17,19 Con-
sumption of any part of the fish, including the entrails and
skin, may result in SPD. Supermarket-bought salmon may also
be infected.17 Infection has also been reported after swimming
activity, without apparent exposure to fish.17
Clinical Features
Signs and Their Pathogenesis
N. helminthoeca replicates within macrophages. This results in a
granulomatous inflammatory response within the stomach, intes-
tines, lymph nodes, and spleen. Granulomatous meningitis has
also been detected in dogs with SPD.20,21 The incubation period
ranges from 2 to 14 days (median, 7 days), but may be as long as
33 days.22 The severity of illness varies considerably (Box 31-1).
Dogs with peracute infection may be found dead.17 A marked
febrile response is commonly detected (>70% of cases) (Box
31-2) and may reach 107.6°F (42°C).17A median rectal tempera-
ture of 105°F (40.6°C) was reported in one series of naturally
B
FIGURE 31-3  A, “Salt grain” appearance of metacercariae of Nanophyetus salmincola
(arrows) in the kidneys of a Chinook salmon. (Courtesy Craig Banner, Oregon Department
of Fish and Wildlife.) B, Wet mount squash of fresh kidney material from a Chinook salmon
showing a metacercaria of N. salmincola. Bar = 100 µm. (Courtesy Dr. Ronald Hedrick, Uni-
versity of California, Davis.)
BOX 31-1
Signs in the Clinical History of 29 Dogs with Salmon
Poisoning Disease
Inappetence or anorexia: 100%
Lethargy: 86%
Vomiting: 86%
Diarrhea: 72%
Hematochezia: 38%
Weight loss: 28%
Melena: 21%
Polyuria and polydipsia: 3%
infected dogs.17 Terminally ill dogs may be hypothermic. Vir-
tually all dogs develop inappetence or anorexia, and lethargy
is also common. Vomiting occurs in more than 80% of dogs,
and rarely, hematemesis is present.17 Diarrhea is also common
(>70% of dogs) and may be semiformed to liquid in consistency,
sometimes containing frank blood, melena, and, less commonly,
mucus.17 Occasionally, a history of diarrhea and vomiting is
absent. Other signs include weight loss and increased thirst and
urination. Neurologic signs that include mental obtundation,
myoclonic twitching, seizures, and apparent neck pain have been
reported in fewer than 20% of affected dogs, a primary differen-
tial diagnosis being canine distemper virus infection.17
Physical Examination Findings
Dehydration is frequently noted on physical examination
and may be severe. More than 70% of dogs have peripheral
 CHAPTER 31  Salmon Poisoning Disease 313

lymphadenomegaly, which may be generalized or involve only
some peripheral lymph nodes (see Box 31-2). Lymph nodes may
be up to 4 cm in diameter and firm on palpation. Lymphad-
enomegaly may be absent occasionally, sometimes as a result
of lymph node necrosis. Abdominal palpation may reveal pain,
and splenomegaly may also be detected. Other abnormalities
include tachypnea or labored respirations, tachycardia, scleral
or conjunctival injection, and edema that involves the limbs,
face, or cervical region.17 Mucosal pallor, tachycardia, and
weak pulses may be present in dogs with hypovolemic shock.
Cardiac arrhythmias such as ventricular premature contractions
have also been noted, and there may be signs of hemorrhage
such as epistaxis and hyphema. Rectal examination may indi-
cate the presence of liquid stool, sometimes accompanied by
fresh blood or melena.
Diagnosis
Laboratory Abnormalities
Complete Blood Count
Thrombocytopenia is present in 90% of affected dogs. Ane-
mia is present in up to 40% of dogs with SPD and generally
occurs as a result of gastrointestinal hemorrhage. More com-
monly, the CBC shows evidence of lymphopenia, thrombo-
cytopenia, or neutrophilia, frequently with bandemia (Table
31-1). Uncommonly, neutropenia occurs.17 Toxic changes
may be present within neutrophils, and immature monocytes
may occasionally be found in the peripheral blood. Despite
the association with helminthiasis, eosinophilia has not been
documented.
Serum Chemistry Profile
Hyponatremia and hypokalemia are the most common elec-
trolyte abnormalities in dogs with SPD and result from gas-
trointestinal losses that occur with vomiting and diarrhea
(Table 31-2). Low ionized calcium concentrations may be
detected in some dogs, possibly as a result of decreased intes-
tinal absorption. Hypoalbuminemia is present in most (>80%)
dogs. Hypoglobulinemia and hypocholesterolemia occur in
fewer than 25% of dogs with SPD, but may become pro-
found. Abnormal liver enzyme activities may be present, the
most common of which is increased ALP activity,17,20 but
increases in the activity of ALT and AST may also occur.
Hyperbilirubinemia is present in fewer than 20% of dogs. Azo-
temia can be evident in dogs with severe dehydration.

BOX 31-2 Urinalysis

Bilirubinuria and proteinuria are frequently detected on urinaly-
Physical Examination Findings in 29 Dogs with Salmon sis in dogs with SPD.17 Uncommonly, cylindruria and glucos-
Poisoning Disease uria are present, perhaps as a result of renal injury secondary
to impaired renal perfusion. Microscopic hematuria occurs in
some dogs, possibly reflecting underlying coagulopathies that
Peripheral lymphadenomegaly: 74%
can occur in dogs with SPD.
Fever: 73%
Dehydration: 65%
Abdominal pain: 28%
Clotting Function
Evaluation of clotting function in some dogs with SPD shows
Tachypnea or labored respirations: 24%
evidence of disseminated intravascular coagulation. Abnormali-
Tachycardia: 24%
ties include increased PT or APTT, increased fibrin degradation
Neurologic signs: 17%
products, and decreased antithrombin activity.17
Weak pulses: 17%
Splenomegaly: 14% Diagnostic Imaging
Scleral injection: 14%
Mucosal pallor: 10%
Plain Radiography
Abdominal radiographs may be unremarkable or reveal fluid-
Hypothermia: 5%
filled small intestines; decreased serosal detail; and less fre-
quently, hepatomegaly or splenomegaly. Thoracic radiographs

TABLE 31-1
Hematologic Findings in 29 Dogs with Salmon Poisoning Disease
Percent below Percent within Percent above Range for Dogs Number
Reference the Reference the Reference the Reference with Salmon of Dogs
Test Range Range Range Range Poisoning Disease Tested
Hematocrit (%) 40-55 39 61 0 22-53 23
Neutrophils (cells/µL) 3000-10,500 4 53 43 728-26,224 23
Band neutrophils (cells/µL) Rare 0 40 60 0-6825 20
Monocytes (cells/µL) 150-1200 9 61 30 42-3290 23
Lymphocytes (cells/µL) 1000-4000 78 22 0 0-2070 23
Eosinophils (cells/µL) 0-1500 0 100 0 0-690 21
Platelets (cells/µL) 150,000-400,000 90 0 10 10,000-446,000 20
314 SECTION 2  Bacterial Diseases

TABLE 31-2
Findings on Serum Biochemistry Analysis in 29 Dogs with Salmon Poisoning Disease
Percent below Percent within Percent above Range for Dogs
Reference the Reference the Reference the Reference with Salmon Number of
Test Range Range Range Range Poisoning Disease Dogs Tested
Sodium (mmol/L) 145-154 59 36 5 133-161 22
Potassium (mmol/L) 4.1-5.3 41 59 0 3.7-5.0 22
Chloride (mmol/L) 105-116 9 77 14 98-118 22
Bicarbonate (mmol/L) 16-26 20 15 65 11-28 20
Calcium (mg/dL) 9.9-11.4 83 17 0 7.2-10.9 23
Phosphorus (mg/dL) 3.0-6.2 9 74 17 2.7-13.0 23
Creatinine (mg/dL) 0.5-1.6 4 4 13 0.4-3.3 23
BUN (mg/dL) 8-31 0 83 17 8-110 23
Albumin (g/dL) 2.9-4.2 83 17 0 0.9-3.8 23
Globulin (g/dL) 2.3-4.4 26 70 4 1.6-4.7 23
Cholesterol (mg/dL) 135-345 22 69 9 79-408 23
Total bilirubin (mg/dL) 0-0.4 0 83 17 0-3.1 23
ALT (U/L) 19-70 4 74 22 17-1,443 23
ALP (U/L) 15-127 0 39 61 29-985 23

loops, sometimes with wall thickening, corrugation, hyper-

motility, or hypomotility, may be documented. Uncommonly,
hepatomegaly or a small amount of free peritoneal fluid is
identified.

Microbiologic Tests
Diagnostic assays for SPD are summarized in Table 31-3.

Diagnosis Using Fecal Examination

FIGURE 31-4  Enlarged and hypoechoic abdominal (medial iliac) lymph nodes as
detected using abdominal sonographic examination in a 9-year-old male German shep-
herd dog with salmon poisoning disease.
are typically unremarkable or show reduction in size of the
intrathoracic vasculature, consistent with hypovolemia.
Sonographic Findings
Abdominal ultrasound examination of dogs with SPD often
shows mild or moderate generalized abdominal or mesenteric
lymphadenomegaly, as well as splenomegaly with a mottled
splenic echotexture. Some dogs that lack peripheral lymphad-
enomegaly have abdominal lymphadenomegaly on ultrasound
examination,17 and ultrasound-guided aspiration of enlarged
abdominal lymph nodes can be helpful for diagnosis in dogs
that lack peripheral lymphadenomegaly. Lymph nodes develop
a rounded appearance with a hypoechoic or hypoechoic
mottled echogenicity (Figure 31-4). Fluid-distended intestinal
SPD can be diagnosed when operculated trematode eggs are
found in fecal specimens of dogs with consistent clinical signs,
which appear within 5 to 8 days after the ingestion of infected
fish. Although the sensitivity of centrifugal zinc sulfate fecal
flotation for detection of N. salmincola ova in dogs with SPD
is similar to that of fecal sedimentation, some dogs that test
negative by one method test positive using the other, so the sen-
sitivity of fecal examination for diagnosis is maximized by per-
forming both centrifugal zinc sulfate fecal flotations and fecal
sedimentation together. In one study, 93% of dogs with SPD
tested positive for N. salmincola ova using a combination of
fecal flotation and fecal sedimentation.17 False negatives may
occur when the duration of illness is shorter than the prepatent
period of N. salmincola or when light fluke burdens are pres-
ent. The finding of fluke ova on fecal examination may not be
diagnostic of SPD, because dogs may be infected with trema-
todes that do not harbor the rickettsia, and ova may be shed
for months after recovery. Nevertheless, N. salmincola ova were
detected in only 0.2% of more than 1800 fecal flotations in dogs
seen at a teaching hospital in an endemic area, and all positive
test results were in dogs suspected to have SPD. Thus, the speci-
ficity of fecal examination for a diagnosis of SPD appears to be
extremely high.17
Centrifugal zinc sulfate fecal flotation may reveal co-infec-
tions with other gastrointestinal parasites, which can complicate
 CHAPTER 31  Salmon Poisoning Disease 315

the clinical picture. Co-infections with Dipylidium caninum and dogs.17 Real-time PCR assays for N. helminthoeca are avail-
Trichuris vulpis have been reported in dogs with SPD.17 able from some veterinary molecular diagnostic laboratories
and can be performed on blood, lymph-node, or splenic aspi-
Cytologic Diagnosis rates and possibly fecal specimens from dogs with SPD in
In addition to fecal examination for trematode ova, SPD can be order to confirm the diagnosis. More research is required to
diagnosed following cytologic examination of peripheral lymph understand the sensitivity and specificity of PCR assays for
node aspirates, or ultrasound-guided aspirates of abdominal diagnosing SPD in dogs, including the optimum specimen type
lymph nodes or the spleen. Cytologic findings consist of histio- for testing.
cytic hyperplasia and lymphoid reactivity. Rickettsial organ-
isms are 0.3−µm cocci or coccobacilli and may be seen in large
numbers within the cytoplasm of macrophages. They resemble
granular to amorphous material of a uniform blue color using
Wright’s stain, occasionally forming loose clusters of organ-
isms (morulae) (Figure 31-5).17,22,23 In as many as one quarter
to one third of dogs, rickettsial organisms are not identified.
The presence of histiocytic hyperplasia should alert the clini-
cian to the possibility of SPD. Organisms may also be absent in
dogs with a recent history of antimicrobial therapy.

Serologic Diagnosis
Serologic tests for N. helminthoeca antibodies or antigen are
not available on a commercial basis for veterinary practitio-
ners. Antibodies to N. helminthoeca can cross-react sero-
logically with those of Ehrlichia spp., so it is possible that
dogs with SPD might test positive using serologic tests for
Ehrlichia spp. (provided enough time had elapsed for anti-
body formation to occur).24 In one study, most dogs with
SPD that were tested for antibodies against other rickettsial
pathogens did not seroreact to Ehrlichia canis or Anaplasma
phagocytophilum.17

Molecular Diagnosis Using the Polymerase Chain Reaction FIGURE 31-5  Lymph node aspirate from a dog with salmon poisoning disease. Note
Specific PCR assays have been used to detect the DNA of the large histiocyte that contains granular coccoid organisms consistent with Neorickettsia
Neorickettsia helminthoeca in clinical specimens from affected helminthoeca.

TABLE 31-3
Diagnostic Assays Available for Salmon Poisoning Disease in Dogs
Assay Specimen Type Target Performance
Fecal sedimentation Feces Nanophyetus salmincola Specificity nears 100% when performed by a parasi-
combined with zinc ova tologist. Sensitivity probably >90%. Eggs are light
sulfate centrifugal brown, ovoid, operculate at one end and measure
fecal flotation 0.087 to 0.097 mm × 0.038 to 0.055 mm.
Cytology Lymph-node or Detection of N
­ eorickettsia Sensitivity >70% when performed by a veterinary
splenic aspirates helminthoeca within clinical pathologist. Sensitivity may be improved by
­macrophages examination of aspirates from multiple lymph node.
Organisms within macrophages may be confused
with debris or hemosiderin. Other changes include
lymphocytic reactivity and histiocytic hyperplasia.
Histopathology Biopsy or ­necropsy N. helminthoeca within Organisms may be stained with Giemsa. Antimicro-
specimens, espe- ­macrophages bial therapy can lead to false-negative results.
cially lymph nodes,
spleen, gastrointes-
tinal tissues
PCR Blood, lymph- N. helminthoeca DNA Well validated assays that are specific for
node or splenic N. ­helminthoeca are not available commercially.
aspirates, tissue Optimum specimen type unknown. Antimicrobial
specimens, feces therapy may lead to negative PCR results.
316 SECTION 2  Bacterial Diseases

Pathologic Findings
Gross Pathologic Findings
Gross necropsy findings in dogs with SPD consist of enlarge-
ment of lymphoid tissues, including the tonsils, thymus,
lymph nodes, and spleen. Lymph nodes may be yellowish and
edematous and may contain white foci that represent foci of
granulomatous inflammation. Petechial hemorrhages may be
noted on the lymph nodes, the gastrointestinal tract, pan-
creas, gallbladder, and urinary bladder. The gastrointestinal
tract may be thickened and contain white foci. The lumen of
the intestinal tract may contain free blood (Figure 31-6, A).
Flukes are only 0.8 to 1.1 mm long and 0.3 to 0.5 mm wide25
and may not always be visible grossly.

Histopathologic Findings A
Lymphoid tissues show marked depletion of lymphoid fol-
licles, and subcapsular and medullary sinuses are filled with
histiocytes, the cytoplasm of which can contain numerous
rickettsial organisms, which occasionally form clusters within
vacuoles. Organisms are readily appreciated using Giemsa
stain (see Figure 31-6, C). Lymph node and splenic necrosis
may also be present.17,22 Inflammatory nodules that contain
lymphocytes, plasma cells, and infected histiocytes expand the
lamina propria of the gastrointestinal tract and extend into
the submucosa. Parasites consistent with N. salmincola may
be found embedded in the mucosa, primarily within the duo-
denum (see Figure 31-6, B).16,21 Nevertheless, flukes are not
always found using histopathology in dogs that die from SPD,
even in the absence of anthelmintic therapy.
One dog that died of SPD had widespread and multiple
thromboses involving numerous organs with associated tis- B
sue infarction. Granulomatous meningitis has been described
at necropsy in dogs that were experimentally infected with
N. helminthoeca.20

Treatment and Prognosis

The treatment of choice for SPD is doxycycline, tetracycline,
or oxytetracycline, which should be given for a minimum of
7 days. Administration of tetracyclines is generally associated
with clinical improvement within 24 hours, and signs resolve
within 1 to 4 days.17 Dogs that are vomiting may require
treatment with parenteral tetracyclines. Clinical improvement
has also been reported following treatment with enrofloxacin
and parenteral trimethoprim-sulfamethoxazole. Antimicrobi-
als that do not appear to be effective include first-generation
cephalosporins, penicillins, aminoglycosides, chlorampheni-
col, and metronidazole.17 Although complete clinical recovery
C
may occur without anthelmintic treatment, the fluke infection FIGURE 31-6  A, Intestinal mucosa of an 8-month-old female spayed Maltese mix dog
should be treated with praziquantel after initial recovery from that died of salmon poisoning disease. The intestinal wall was thickened and the contents were
SPD (Table 31-4). dark red and mucoid. (Courtesy University of California, Davis Veterinary Anatomic Pathology
Dogs with mild signs or infrequent vomiting may respond Service.) B, Duodenum. A transverse section of an adult Nanophyetus fluke is embedded
well to treatment with oral tetracyclines alone, whereas those within the intestinal villi. H&E stain. Bar = 200 µm. C, Lymph node, medullary sinus from a dog
with salmon poisoning disease. Many histiocytes have intracytoplasmic clusters of coccobacilli
with more severe disease often require hospitalization for IV
less than 1 µm in diameter. Giemsa. Bar = 5 µm. (B and C from Sykes JE, Marks SL, Mapes S,
antimicrobial and crystalloid fluid therapy (see Table 31-4). et al. Salmon poisoning disease in dogs: 29 cases. J Vet Intern Med 2010;24[3]:504-513, 2010.)
Oral antimicrobial therapy can be continued once vomiting
ceases. The presence of severe hypovolemia or hypoalbumin-
emia may necessitate treatment with colloids such as fresh such as metoclopramide, maropitant, or ondansetron. Close
frozen plasma, dextrans, or hetastarch. Packed RBC or whole monitoring of hematocrit, albumin, electrolytes, acid-base
blood may be required for dogs with severe anemia. Treatment status, renal parameters, and coagulation parameters may be
with total or partial parenteral nutrition may also be required required in some severely affected dogs. Blood cultures should
if vomiting is persistent and does not respond to antiemetics be considered in dogs with severe hemorrhagic diarrhea and

TABLE 31-4
Antimicrobials Used to Treat Salmon Poisoning Disease in Dogs
Dose Interval Duration
Drug (mg/kg) Route (hours) (days)
Doxycycline 5 PO, IV 12 7-14
Tetracycline 22* PO 8 7
7 IV 8 7
Oxytetracycline 7-10* PO, IV 8 7
Praziquantel† 10-30 PO 24 1-2
*Reduce dose in renal failure; may cause teeth discoloration in young
animals. Monitor for nephrotoxicity (cylindruria).
†For treatment of fluke infection.
abnormal perfusion parameters, after which treatment with
broad-spectrum parenteral antimicrobial drugs should be
commenced.
Prolonged hospitalization may be required for dogs that
develop complications such as cardiac arrhythmias, intus-
susceptions, adverse reactions to tetracyclines, disseminated
intravascular coagulation, or septicemia.17,19 In a study of dogs
evaluated at a tertiary referral hospital, death or euthanasia
occurred in 14% of 29 dogs with SPD.17 The mortality may be
lower in dogs seen in primary care practice, providing appropri-
ate treatment can be administered. In general, early treatment
is associated with an excellent prognosis. Without treatment,
death can occur within 5 to 10 days.
CASE EXAMPLE
Signalment: “Herman,” 9-year-old male German shepherd
dog from Sacramento, CA (Figure 31-7).
History: Herman’s owner reported a 2-day history of
inappetence, lethargy, and one episode of vomiting. The dog
had been drinking normally. There had been no diarrhea,
coughing, sneezing, or increased thirst and urination. The
owner also reported that Herman ate turkey and chicken
that contained bones 3 days before the onset of illness. He
was normally fed a strict diet of rabbit- and potato-based
prescription dog food and grilled salmon because of atopic
dermatitis. Herman was sometimes fed salmon scraps from
the local supermarket, which the owner grilled briefly. He
had not traveled out of his local area. He was up to date
on vaccinations, which included vaccines for distemper,
hepatitis, parvovirus, and rabies.
Current Medications: Lincomycin 29 mg/kg PO q12h;
trimeprazine tartrate 0.2 mg/kg PO q48h; prednisolone 0.1
mg/kg q48h for atopic dermatitis.
Other Medical History: Herman had been diagnosed with
hip osteoarthritis using pelvic radiography 8 months earlier.
Controlled weight loss was the recommended treatment.
CHAPTER 31  Salmon Poisoning Disease 317
Immunity and Vaccination
Animals that recover from SPD are immune to reinfection with
the same strain of N. helminthoeca,22,26,27 but challenge with
an alternate strain (then the EFF agent) results in disease, and
antibodies fail to cross react with the alternate strain. This may
explain why SPD has been reported to occur more than once in
some dogs.17,22 No vaccine is available.
Prevention
N. helminthoeca metacercariae are effectively destroyed by proper
cooking or freezing of infected fish. Ingestion of raw fish by dogs in
endemic areas should be discouraged, and dog owners in endemic
areas should be educated about the disease. Dogs that have eaten
raw fish from an endemic area, or those swimming in rivers or
lakes in these areas, should be watched carefully for signs of leth-
argy, vomiting, or decreased appetite that occur within 2 weeks of
exposure. If multiple dogs are simultaneously exposed to a source
of N. helminthoeca, and one dog develops SPD, treatment of the
other dogs in the group with 7 days of doxycycline should be con-
sidered. Medical equipment used in the treatment of dogs with SPD
should not be reused on other patients without proper sterilization.
Public Health Aspects
Human infection with N. helminthoeca has not been reported.
However, humans may become infected with the fluke, N. sal-
mincola, after consumption of poorly cooked fish. Most humans
are subclinically infected, although abdominal discomfort, diar-
rhea, vomiting, weight loss, nausea, and peripheral eosinophilia
may occur.28
He was cryptorchid, and the abdominal testicle had been
removed when he was a young dog. The descended testicle
was still present.
Physical Examination:
Body Weight: 51.5 kg
General: Quiet, alert and responsive. Ambulatory on all four
limbs. T = 104.3°F (40.2°C), HR = 108 beats/min, RR = 60
breaths/min, mucous membranes pink, CRT = 1 s.
Integument: A full, shiny haircoat was present and there was
no evidence of ectoparasites.
Eyes, Ears, Nose, and Throat: No significant abnormalities
were noted. A mild amount of brown waxy debris was
present within both ear canals.
Musculoskeletal: A body condition score of 6/9 with
symmetrical muscling was noted. Mild to moderate pelvic
limb weakness was identified.
Cardiovascular: Strong femoral pulses were noted. No murmurs
or arrhythmias were detected on thoracic auscultation.
Respiratory: An increased respiratory rate with increased
abdominal effort was present.
Gastrointestinal and Genitourinary: Abdominal palpation
revealed a tense abdomen, with abdominal splinting, and
splenomegaly. There was one descended testicle, which was
smooth with no masses. No abnormalities were detected on
Continued
318 SECTION 2  Bacterial Diseases
FIGURE 31-7  “Herman,” a 9-year-old male German shepherd dog with salmon
­poisoning disease.
rectal examination. Soft feces were present in the rectum,
and the prostate was palpable and symmetrical.
Lymph Nodes: Marked peripheral lymphadenopathy was
present. Mandibular, superficial cervical, and popliteal
lymph nodes were all 3-4 cm in diameter and firm.
Laboratory Findings:
CBC:
HCT 40.4% (40-55%)
MCV 65 fL (65-75 fL)
MCHC 36.6 g/dL (33-36 g/dL)
WBC 4240 cells/µL (6000-13,000 cells/µL)
Neutrophils 3689 cells/µL (3000-10,500 cells/µL)
Lymphocytes 466 cells/µL (1000-4000 cells/µL)
Monocytes 42 cells/µL (150-1200 cells/µL)
Platelets 74,000 platelets/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 141 mmol/L (145-154 mmol/L)
Potassium 4.0 mmol/L (3.6-5.3 mmol/L)
Chloride 112 mmol/L (108-118 mmol/L)
Bicarbonate 13 mmol/L (16-26 mmol/L)
Phosphorus 2.8 mg/dL (3.0-6.2 mg/dL)
Calcium 9.9 mg/dL (9.7-11.5 mg/dl)
BUN 15 mg/dL ( 5-21 mg/dL)
Creatinine 1.1 mg/dL (0.3-1.2 mg/dL)
Glucose 86 mg/dL (64-123 mg/dL)
Total protein 6.9 g/dL (5.4-7.6 g/dL)
Albumin 3.6 g/dL (3.0-4.4 g/dL)
Globulin 3.3 g/dL (1.8-3.9 g/dL)
ALT 108 U/L (19-67 U/L)
AST 109 U/L (19-42 U/L)
ALP 105 U/L (21-170 U/L)
Creatine kinase 381 U/L (51-399 U/L)
GGT < 3 U/L (0-6 U/L)
Cholesterol 296 mg/dL (135-361 mg/dL)
Total bilirubin 0.4 mg/dL (0-0.2 mg/dL)
Magnesium 1.7 mg/dL (1.5-2.6 mg/dL).
Urinalysis: SGr 1.048; pH 6.0, 2+ protein (SSA), 1+ bilirubin, 1+
hemoprotein, no glucose, 0-1 WBC/HPF, 20-30 RBC/HPF, rare
ammonium biurate crystals, many lipid droplets
Imaging Findings:
Thoracic Radiographs: The cardiovascular and pulmonary
structures appeared within normal limits. A mild diffuse
bronchial pattern throughout all lung fields was considered
consistent with the age of the patient. Marked spondylosis
deformans was present throughout the thoracic spine.
Abdominal Ultrasound: The liver had a diffuse, coarse
echotexture. The spleen was markedly, diffusely enlarged
and had a diffuse hypoechoic, mottled echotexture. The
prostate gland was enlarged and had multiple small
anechoic cysts within its parenchyma. One testicle was
present in the scrotum and had a striated, heterogenous
echotexture. A second inguinal or intra-abdominal testis
was not observed. Multiple enlarged, hypoechoic inguinal
lymph nodes were present (see Figure 31-3). A large (2.5 cm),
round, hypoechoic mass with a focal area of mineralization
was present in the cranial abdomen caudal to the liver and
most likely represented a lymph node. Multiple enlarged,
hypoechoic sublumbar lymph nodes were observed. The
splenic lymph nodes were enlarged and mildly hypoechoic.
Cytology Findings: Cytology of ultrasound-guided splenic
aspirate: splenic stromal clumps contained increased
numbers of plump histiocytes and many large clumps of
hemosiderin. Histiocytes were also present in moderate
numbers throughout the smear, often in clumps, and often
mildly vacuolated. They were occasionally erythrophagocytic
and often contain hemosiderin. Interpretation: moderate to
marked histiocytic inflammation.
Cytology of popliteal lymph node aspirate (see Figure 31-5):
moderate reactive lymphoid hyperplasia with pyogranu-
lomatous inflammation was identified. Rare histiocytes
were present that contained intracellular small coccoid
purple-blue structures that were organized individually
and in clusters. These were consistent with Neorickettsia
helminthoeca organisms.
Fecal examination: Fecal flotation: Negative for parasites.
Fecal sedimentation: Nanophyetus salmincola ova, <1 per 10× field
Diagnosis: Salmon poisoning disease
Treatment: Doxycycline, 5 mg/kg PO q12h for 14 days. This
was associated with resolution of fever and inappetence
within 24 hours of starting treatment. By 7 days after
diagnosis, Herman’s owner reported that Herman was
completely back to his normal self.
Comments: This dog was diagnosed with SPD based on the
presence of characteristic cytologic findings on examination
of a lymph node aspirate, together with the detection of N.
salmincola ova in the stool. The case was unusual because
the dog was infected after ingestion of incompletely cooked
supermarket-bought salmon, which the dog had been fed
for years without developing illness. Although the splenic
aspirate contained inflammatory cells typical of those seen
in SPD, organisms could not be convincingly documented.

SUGGESTED READINGS
Mack RE, Bercovitch MG, Ling GV, et  al. Salmon poisoning disease
complex in dogs—a review of 45 cases. Calif Vet. 1990;44:42-45.
Sykes JE, Marks SL, Mapes S, et al. Salmon poisoning disease in dogs:
29 cases. J Vet Intern Med. 2010;24(3):504-513.
REFERENCES
1. Millemann RE, Knapp SE. Biology of Nanophyetus salmincola and
“salmon poisoning disease.” In: Dawes B, ed. Advances in Parasi-
tology. vol. 8. New York, NY: Academic Press; 1970:1-39.
2. Harrell LW, Deardoff TL. Human nanophyetiasis: transmission by
handling naturally infected coho salmon (Oncorhynchus kisutch).
J Infect Dis. 1990;161(1):146-148.
3. Foreyt WJ, Thorson S, Gorham JR. Experimental salmon poi-
soning disease in juvenile coyotes (Canis latrans). J Wildl Dis.
1982;18(2):159-162.
4. Hedrick RP, Arnandi A, Manzer D. Nanophyetus salmincola: the
trematode vector of Neorickettsia helmintheca. California Veteri-
narian. 1990;44:18-22.
5. Gai JJ, Marks SL. Salmon poisoning disease in two Malayan sun
bears. J Am Vet Med Assoc. 2008;232(4):586-588.
6. Headley SA, Scorpio DG, Barat NC, et  al. Neorickettsia hel-
minthoeca in dog. Brazil. Emerg Infect Dis. 2006;12:1303-1304.
7. Headley SA, Kano FS, Scorpio DG, et  al. Neorickettsia hel-
minthoeca in Brazilian dogs: a cytopathological, histopatho-
logical and immunohistochemical study. Clin Microbiol Infect.
2009;15(suppl 2):21-23.
8. Cordy DR, Gorham JR. The pathology and etiology of salmon dis-
ease in the dog and fox. Am J Pathol. 1950;26:617-637.
9. Farrell RK. Transmission of two rickettsia-like disease agents of
dogs by endoparasites in northwestern USA. Proc 1st Int Congr
Parasitol. 1966:438.
10. Farrell RK, Leader RW, Johnston SD. Differentiation of salmon
poisoning disease and Elokomin fluke fever: studies with the black
bear (Ursus americanus). Am J Vet Res. 1973;34:919-922.
11. Kitao T, Farrell RK, Fukuda T. Differentiation of salmon poisoning
disease and Elokomin fluke fever: fluorescent antibody studies with
Rickettsia sennetsu? Am J Vet Res. 1973;34:927-928.
12. Frank DW, McGuire TC, Gorham JR, et  al. Cultivation of
two species of Neorickettsia in canine monocytes. J Infect Dis.
1974;129:257-262.
CHAPTER 31  Salmon Poisoning Disease 319
13. Bennington E, Pratt I. The life history of the salmon-poisoning fluke,
Nanophyetus salmincola (Chapin). J Parasitol. 1960;46(1):91-100.
14. Foreyt WJ, Gorham JR, Green JS, et al. Salmon poisoning disease
in juvenile coyotes: clinical evaluation and infectivity of metacer-
cariae and rickettsiae. J Wild Dis. 1987;23(3):412-417.
15. Baldwin NL, Milleman RE, Knapp SE. “Salmon poisoning” dis-
ease. 3. Effect of experimental Nanophyetus salmincola infection
of the fish host. J Parasitol. 1967;53(3):556-564.
16. Bosman DD, Farrell RK, Gorham JR. Non-endoparasite transmis-
sion of salmon poisoning disease of dogs. J Am Vet Med Assoc.
1970;156:1907-1910.
17. Sykes JE, Marks SL, Mapes S, et al. Salmon poisoning disease in
dogs: 29 cases. J Vet Intern Med. 2010;24(3):504-513.
18. Farrell RK, Dee JF, Ott RL. Salmon poisoning in a dog fed kippered
salmon. J Am Vet Med Assoc. 1968;152:370-371.
19. Mack RE, Bercovitch MG, Ling GV, et al. Salmon poisoning disease
complex in dogs—a review of 45 cases. Calif Vet. 1990;44:42-45.
20. Philip CB, Hadlow WJ, Hughes LE. Neorickettsia helminthoeca,
a new rickettsialike disease agent of dogs in western United
States transmitted by a helminth. Proc 6th Int Congr Microbiol.
1953;4:70-82:Rome.
21. Hadlow WJ. Neuropathology of experimental salmon poisoning of
dogs. Am J Vet Res. 1957;18:898-908.
22. Gorham JR, Foreyt WJ. Salmon poisoning disease. In: Greene CE,
ed. Clinical Microbiology and Infectious Diseases of the Dog and
Cat. 1st ed. Philadelphia, PA: WB Saunders; 1984:538-544.
23. Johns JL, Strasser JL, Zinkl JG, et al. Lymph node aspirate from a
California wine-country dog. Vet Clin Pathol. 2006;35:243-246.
24. Rikihisa Y. Cross-reacting antigens between Neorickettsia hel-
minthoeca and Ehrlichia species, shown by immunofluorescence and
Western immunoblotting. J Clin Microbiol. 1991;29:2024-2029.
25. Witenburg G. On the anatomy and systematic position of the caus-
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26. Donham CR, Simms BT, Miller FW. So-called salmon poisoning in
dogs (progress report). J Am Vet Med Assoc. 1926;68:701-715.
27. Sakawa H, Farrell RK, Mori M. Differentiation of salmon poison-
ing disease and Elokomin fluke fever: complement fixation. Am
J Vet Res. 1973;34:923-925.
28. Eastburn RL, Fritsche TR, Terhune Jr CA. Human intestinal
infection with Nanophyetus salmincola from salmonid fishes. Am
J Trop Med Hyg. 1987;36(3):586-591.
CHAPTER 32
Coxiellosis and Q Fever
Jane E. Sykes and Jacqueline M. Norris
Overview of Coxiellosis and Q Fever
First Described: mid 1930s, Queensland, Australia, in slaugh-
terhouse workers (Derrick)1
Cause: Coxiella burnetii, an obligately intracellular gram-neg-
ative bacteria that belongs to the Family Coxiellaceae
Affected Hosts: Humans are most susceptible; reproductive
disease can occur in farm animal species such as sheep,
goats, cattle, and horses, as well as cats and dogs
Geographic Distribution: Worldwide, with the exception of
New Zealand
Major Clinical Signs: Dogs and cats are usually subclinically
infected. Abortion, premature birth, and stillbirth may
occur.
Differential Diagnoses: Brucellosis, bartonellosis, herpesviral
infections, leptospirosis, neosporosis, canine distemper,
and canine parvovirus infection. Noninfectious causes of
pregnancy loss such as endocrinopathies, genetic disor-
ders, and nutritional deficiency should also be considered.
Human Health Significance: Q fever in people results primar-
ily from contact with infected sheep, goats, and cattle, or
indirect contact with surfaces contaminated with secre-
tions or excretions of these animals. However, contact
with infected companion animals can also lead to trans-
mission. Q fever in people is most commonly an acute
febrile illness, but pneumonia and chronic complica-
tions such as endocarditis, granulomatous hepatitis, and
chronic fatigue can occur.
Etiology and Epidemiology
Coxiella burnetii is a gram-negative, obligately intracellular,
small, pleomorphic rod that causes Q fever in humans, a signifi-
cant zoonosis worldwide. Sheep, cattle, and goats are the most
commonly reported reservoirs for the organism, but infected
cats and less commonly dogs have also been implicated in trans-
mission to people.2-5 Animals other than humans are often sub-
clinically infected, although reproductive complications such as
premature birth, abortion, and stillbirth may also occur. Res-
ervoir hosts shed organisms in urine, feces, saliva, and/or milk,
and especially in vaginal secretions and the products of partu-
rition because of recrudescence of infection during pregnancy.
The placentas of infected animals are heavily infected (up to
109 organisms per gram of tissue). At parturition, aerosols are
created, which are subsequently inhaled by other animals and
humans (Figure 32-1). Ingestion of unpasteurized contaminated
320
milk is a less common and more controversial mode of trans-
mission.6,7 The term Q fever stands for query fever, the name it
was given when it first emerged in Queensland, Australia, and
the underlying cause had not yet been identified. In humans,
acute Q fever is characterized by a febrile “flulike” illness, with
headaches, and sometimes pulmonary and hepatic involvement.
However, infection is initially asymptomatic in 60% of those
infected.8 Chronic Q fever can follow acute Q fever or asymp-
tomatic infection and can be characterized by cardiac disease
(endocarditis, pericarditis, myocarditis), fatigue syndromes,
skin rash, and neurologic, pulmonary, and hepatic disease. In
avian species, systemic illness has been associated with infection
by other Coxiella-like organisms.9-11
Unlike other obligate intracellular pathogens, C. burnetii sur-
vives prolonged periods in the environment, where it resists high
temperature, a variety of disinfectants, and ultraviolet light. The
organism exists in two forms: a small cell variant, which sur-
vives in the environment, and a large cell variant, which repli-
cates within cells. Survival of the organism for up to 10 months
on wool at 15°C to 20°C (59°F to 68°F) has been reported, and
it can also survive in tissues that have been stored in formalin
for several months. It is inactivated by 5% hydrogen peroxide.12
Although originally classified within the order Rickettsiales, the
availability of sequence information led to its reclassification as
a gammaproteobacterium in the order Legionellales, which also
includes Legionella species. C. burnetii undergoes phase varia-
tion, which is associated with alterations in its cell wall com-
ponents. The variants have nominally been called phase 1 and
phase 2.13 The changes in cell surface antigens and the creation
of phase variants has clinical implications for serologic testing
due to the production of phase 1 and phase 2 antibodies to the
bacterium at different stages of natural infection (see Diagnosis,
later). The whole genome of C. burnetii has been sequenced.14
C. burnetii is extremely contagious, and only small num-
bers of organisms are required to cause disease in people. The
epidemiology of Q fever varies geographically. Because of its
predilection for domestic ruminant species, Q fever is most com-
monly reported from rural areas. C. burnetii has been detected
within a huge variety of different tick and other arthropod spe-
cies. Within ticks, it replicates in the midgut and it is shed in
large quantities in tick feces. Although ticks have the potential
to transmit the organism to domestic animal species, the most
significant role of ticks appears to be in transmission to wild-
life.15 C. burnetii has been identified in fish, wild birds, rodents,
marsupials, horses, swine, camels, marine mammals, ducks, tur-
keys, geese, cats, dogs, and rabbits. Although the mechanism is
not clearly defined, cats and dogs may become infected through
exposure to infected sheep, cattle, and goats—possibly through
ingestion of unpasteurized milk from these species—and
 CHAPTER 32  Coxiellosis and Q Fever 321

FIGURE 32-1  Transmission of Coxiella burnetii. The organism is most often transmitted to humans when animals give birth. People become infected by inhalation when they directly
contact the products of parturition or as a result of indirect contact with organisms that survive in the environment. Sheep, cattle, and goats are the most common reservoirs of the organ-
ism for human infection, but infected cats and less commonly dogs can also transmit the infection to humans. Ticks also harbor the organism and are thought to maintain transmission
to wildlife species, especially rodents (such as small mice) and rabbits, but also birds. Dogs and cats and humans can also become infected when they contact (or ingest) wildlife species.
Ingestion of infected milk may be a lesser mode of transmission.

through contact with, or predation of, infected wildlife reservoir
hosts such as rodents, birds, and rabbits.
Exposure to stillborn kittens or apparently healthy newborn
kittens has been a risk factor for human infection in Africa,
Japan, Korea, Canada, and the United States. In a study of intact
female cats from Colorado, C. burnetii DNA was not detected
in vaginal swab specimens or uterine biopsies from 50 shelter
cats using a PCR assay, but 4 of 47 uterine biopsies from client-
owned cats were positive.16 Seroprevalence studies suggest that
infection is more common in stray cats and dogs than client-
owned animals, but further research is required to determine
risk factors for exposure in dogs and cats.17,18 No association
has been found between prevalence of Q fever in people and cat
or dog ownership.19 Laboratory-acquired infections have been
described in people, and infection can occur as a result of expo-
sure to straw, manure, or dust, such as that from contaminated
farm vehicles and contaminated clothing. Q fever has emerged
as a disease of U.S. combat arms military personnel who are
deployed to Iraq.20 Studies of a large, 2-year outbreak in the
Netherlands showed that dust-accumulating surfaces contained
higher levels of C. burnetii DNA than vaginal swabs from sheep
and goats.21 The organism can travel for miles as a result of
windborne transmission.
organism appears to modulate apoptosis in order to inhibit
cell death and promote its own replication.23 Subsequently,
infected cells enter the bloodstream. After experimental infec-
tion, bacteremia may be prolonged (up to 1 month) in cats.24
The organism can persist in a variety of tissues, and in human
patients, this contributes to chronic disease manifestations, such
as prolonged fever, endocarditis, vasculitis, chronic fatigue, and
immune-mediated consequences such as demyelinating poly-
radiculoneuritis.25 Appropriate diagnosis and treatment at the
acute stage of clinical illness is important in reducing the risk of
postacute Q-fever sequelae and development of chronic disease.
Reactivation of shedding has been described during pregnancy
in humans.12 Infected cats and dogs usually show no clinical
signs of infection, but stillbirth, abortion, and prolonged vagi-
nal bleeding for 3 weeks before delivery have been reported in
cats.4,26 The organism replicates in trophoblasts, and in experi-
mentally infected goats, massive bacterial replication occurs in
the placenta just before abortion.27 An infected rabbit hound
dog that transmitted C. burnetii to a family in Nova Scotia
gave birth to four puppies, all of which died within 24 hours
of birth.5 The importance of C. burnetii in reproductive tract
disease and chronic disease manifestations such as endocarditis
in cats and dogs requires further investigation.

Clinical Features Diagnosis

Signs and Their Pathogenesis
After inhalation (and to a lesser extent, ingestion), C. burnetii
enters mononuclear phagocytes and survives within the harsh
environment of the phagolysosome.22 Remarkably, the acid
conditions of the lysosome appear to be a requirement for the
metabolic functions of C. burnetii. Replication is prolonged,
and minimal cytopathic effects occur in infected cells. The
Microbiologic Tests
Diagnostic assays available for Q fever are listed in Table 32-1.
Culture
C. burnetii can be isolated in a variety of different cell lines.
It has also been isolated in a cell-free medium that mimics the
phagolysosome environment.28 Because it is highly infectious
322 SECTION 2  Bacterial Diseases
TABLE 32-1
Diagnostic Assays Available for Q Fever
Assay Specimen Type Target
Cell culture Placental ­materials, C. burnetii
vaginal swabs, blood,
urine, feces, milk
Histopathology Placental materials C. burnetii
Serology Serum Antibodies to
phase 1 or
phase 2
C. burnetii
PCR Whole blood, tissue C. burnetii DNA
specimens, vaginal
swabs, milk, feces
CFT, Complement fixation test; IFA, immunofluorescent antibody; OIE, World Organization for Animal Health.
to humans, most laboratories do not have the special facilities
required to isolate C. burnetii. Thus diagnosis of infection is usu-
ally performed using immunohistochemistry, serology, or PCR.
Serologic Diagnosis
The World Organization for Animal Health (OIE) reference test
for serologic diagnosis of C. burnetii infection in animals remains
the complement fixation test (CFT),20 despite the availability of
more sensitive serologic tests. A variety of serologic assays have
been used to detect antibodies to phase 1 or phase 2 C. burnetii
antigens in humans and other animals, including immunofluo-
rescent antibody (IFA) assays, ELISA, and the CFT. The CFT
is considered the least sensitive and specific method. In human
patients, anti–phase 2 antibodies (IgG and IgM) are present in
acute Q fever, whereas anti–phase 1 IgG antibodies predominate
in chronic infection.15,29 In humans, the use of IFA assays has
been recommended for diagnosis of Q fever.12 Acute and con-
valescent serology is required for diagnosis of acute Q fever in
people. It can take at least a month before seroconversion occurs;
titers peak around 8 weeks after infection.15 Because infection is
usually not clinically detected in dogs and cats, serologic assays
have primarily been used in dogs and cats for epidemiologic stud-
ies or as part of import requirements to Q fever–free countries
such as New Zealand. Low-level cross-reactivity with other bac-
teria such as Bartonella spp. and chlamydiae has been described
in some studies, which highlights the need for animal species-
specific cutoff values for all serologic methods.30,31 The concur-
rent performance of serology for Bartonella and Chlamydia (i.e.,
to determine if titers are higher or lower to these organisms than
to Coxiella) may assist in interpretation of the results.
Molecular Diagnosis Using the Polymerase Chain Reaction
Both conventional and real-time PCR assays have been described
for detection of C. burnetii DNA. PCR assays are the diagnostic
Performance
Not widely offered or utilized for routine diagnostic purposes.
Requires special facilities.
Organisms may be seen when present in large numbers and stain
with Gimenez stain. Immunohistochemistry and fluorescence in
situ hybridization can be used to identify the organisms as C.
burnetii.
Acute and convalescent serology is required for diagnosis of acute
infection. At least 1 month may be required for seroconversion.
Cross-reactivity may occur to related bacteria. IFA is considered
the serologic test of choice in human patients. CFT is the OIE
standard in animals but is less sensitive.
Confirms active infection. Sensitivity and specificity may vary de-
pending on stage of infection, assay design, and specimen type.
PCR is most sensitive in acute infections (in the first 2 weeks
of illness in humans), but is usually negative in humans with
chronic infections. Subclinical shedding in animals is variable
but is greatest during parturition.
test of choice for organism detection and have revolutionized
knowledge of the epidemiology of C. burnetii infections. In ani-
mals, PCR assays have primarily been used on a research basis.
Negative results can occur with PCR assays in chronic infec-
tions or even as early as 2 weeks after onset of acute illness
in humans, because of undetectable levels of the infection or
absence of bacteremia. Because C. burnetii is highly infectious
to humans, special handling (BSL 3) conditions are required for
fresh specimens that are submitted for C. burnetii PCR assays
(see Table 1-4). When C. burnetii is suspected (such as in pla-
centas or aborted material), the laboratory should be contacted
in advance, and specimens should be labeled and transported
appropriately.
Pathologic Findings
Little information is available about the pathology of C. bur-
netii infection in dogs and cats. In affected livestock, C. burnetii
causes a necrotizing placentitis with mixed inflammatory cell
infiltrate and nonsuppurative vasculitis. In some cases, small,
basophilic intracytoplasmic organisms can be seen (Figure 32-2).
The organisms stain with Gimenez, Giemsa, and modified acid-
fast stains, but not Gram, silver, or periodic acid–Schiff stains.32
Immunohistochemistry and fluorescent in situ hybridization33
can be used to identify C. burnetii antigen within lesions.
Treatment
Many acute infections in people are self-limiting (or even sub-
clinical), and symptoms respond to nonspecific therapy such
as antipyretics and rehydration. However, because of concerns
that relate to the development of chronic Q fever, specific anti-
microbial therapy is recommended regardless of the severity
of acute Q fever. When antimicrobials agents are required,
doxycycline for 14 to 21 days, azithromycin for 3 to 5 days,

FIGURE 32-2  Histopathology showing severe, acute necrotizing placentitis
in the placenta of a goat from which dead fetuses were removed by caesarian sec-
tion. Trophoblasts are distended with abundant basophilic bacterial organisms
(arrows). The organisms were subsequently identified as Coxiella burnetii with
immunohistochemistry.
or quinolones have been used successfully to treat pneumo-
nia and hepatitis in human patients with C. burnetii infection.
Combination therapy, such as doxycycline, ciprofloxacin, and
rifampin for prolonged periods (e.g., 2 years) or doxycycline
and hydroxychloroquine (which alkalinizes phagolysosomes)
has been required for treatment of Q fever endocarditis, and
valve replacement is frequently needed.12,15 Antibody titers
decrease with successful treatment. Q fever during pregnancy,
which may result in abortion when infection occurs in the first
trimester, is treated with trimethoprim-sulfamethoxazole for
the duration of the pregnancy.34 Treatment of an infected sheep
herd with oxytetracycline on days 100 and 120 of gestation did
not prevent shedding of C. burnetii in milk, vaginal swabs, or
feces as assessed with PCR, nor did it affect the duration of
shedding.35
Public Health Aspects
In humans, asymptomatic infections with C. burnetii are proba-
bly widespread. When it occurs, Q fever is most often a systemic
febrile illness. Because it mimics a variety of other illnesses, and
confirmatory diagnostic tests require a specific index of suspi-
cion by the medical clinician, Q fever is most likely underrecog-
nized. Veterinarians involved in mixed or farm animal practice,
slaughterhouse workers, and workers in the livestock industry
(farmers, shearers, transporters of animals) are most likely to
develop Q fever as a result of direct contact with infected live-
stock. Person-to-person spread of Q fever is rarely reported, but
humans can shed organisms during parturition, and infection
of an obstetrician during delivery was described.36 Transmis-
sion during postmortem examinations has also been reported.
Between 2005 and 2008, approximately 140 to 170 cases of
Q fever were reported to the Centers for Disease Control and
CHAPTER 32  Coxiellosis and Q Fever 323
Prevention each year, and rates of hospitalization for these cases
were around 50%.37 However, severe cases are more likely to be
reported, so actual hospitalization rates are likely much lower.
In Australia, 300 to 400 cases are reported annually.38
In humans, acute Q fever occurs after an incubation period
that ranges from 9 to 39 days, although shorter incubation peri-
ods are possible with exposure to high doses of the agent.12 The
incubation period after exposure to an infected cat has ranged
from 9 days to 4 weeks.3,4 Males are predisposed, which relates
to an increased likelihood of exposure. Patients who develop
pneumonia may also develop severe headache, as well as sweats,
chills, fatigue, and myalgia. Other signs include cough, chest
pain, nausea and vomiting, sore throat, diarrhea, a rash, and
abdominal pain and hematuria.4 Myocarditis and pericardi-
tis can occur rarely. Blood work often shows a normal white
cell count, and thrombocytopenia or marked thrombocytosis
can occur. Serum biochemistry testing reveals mild to moder-
ate increases in the activity of hepatic transaminases in almost
all patients.12 Pulmonary infiltrates are present on thoracic
radiographs, and in some cases, pleural effusion.12,15 Q fever
pneumonia is rarely fatal. Chronic Q fever may be manifested
as endocarditis, optic neuritis, osteomyelitis, granulomatous
hepatitis, interstitial pulmonary fibrosis, prolonged fever, and
a chronic fatigue syndrome, which has been described in up to
20% of patients who develop acute infection with C. burnetii.39
Endocarditis follows acute Q fever in fewer than 1% of cases,29
and it may not become apparent for several years after acute
illness. Approximately 3% to 5% of human endocarditis cases
have been attributed to C. burnetii. Host factors seem to be
important in whether chronic manifestations of Q fever occur.
Cellular immunity and the production of IFN-γ appear to be
required for resolution of infection.29
Because treatment with tetracyclines does not seem to reduce
shedding by reservoir hosts, an emphasis has been placed on
the development of effective vaccines for public health pur-
poses. A phase 1 inactivated vaccine (Coxevac) has been used
in Europe on an investigational basis to prevent shedding by
reservoir livestock species, such as sheep and goats. Vaccina-
tion of sheep flocks led to a reduced number of abortions and
decreased shedding, but did not completely prevent infection.29
Vaccination was ineffective in previously infected cattle or naïve
pregnant cattle.40 Another phase 1 whole cell vaccine (Q-VAX,
Commonwealth Serum Laboratories, Parkville, Australia) has
also been used to protect naïve but at-risk humans, such as
livestock workers (farmers, shearers, slaughterhouse workers,
animal transporters), veterinarians, and laboratory workers,
from infection. A Q-VAX skin test as well as serologic assays
are performed as a prescreening of potential vaccine recipients
for prior sensitization to Q fever antigens to minimize adverse
vaccine reactions; humans with prior exposure to C. burnetii
should never be vaccinated. It has been recommended that
humans not drink raw milk from infected farms. When Q fever
is discovered on a farm, sale of milk is forbidden for the year
after initial diagnosis. Aborted material and placentas must be
buried with lime or incinerated.29
324 SECTION 2  Bacterial Diseases

CASE EXAMPLE
Signalments: 50-year-old female veterinarian and a 23-year-
old female veterinary technician from southeastern Australia
History: A 3-year-old female British shorthair cat with dystocia
was brought to a veterinary clinic by the breeder/owner
at 8 o’clock in the morning. One kitten was stillborn in the
home environment overnight. Safe delivery of the two
remaining kittens required a cesarean section. Surgery was
performed immediately, and the kittens were delivered and
revived by vigorous rubbing with dry towels. The kittens
were discolored by yellow-green placental material and
were slow to revive. They were able to successfully to suckle
once the queen had recovered from general anesthesia. The
queen and kittens were returned to the breeder’s cattery the
same afternoon.
Two weeks after the surgery, the veterinarian and the vet-
erinary technician who had been involved in the cesarean
section developed fever, severe headache, neck pain, chills,
night sweats, myalgia, and stiffness. The veterinarian sought
medical attention. Psittacosis was considered the most likely
diagnosis by the attending physician, because psittacine
birds were frequently treated in the clinic. A serologic test
for chlamydiosis was negative, but nevertheless treatment
with doxycycline was commenced. The veterinary techni-
cian’s clinical signs were more severe and included cough,
chest pain, nausea, and vomiting. As a result, she was hospi-
talized for 3 weeks for further diagnostic testing, treatment,
and convalescence. When the technician tested positive by
PCR assay for Coxiella burnetii DNA on day 10 after the onset
of illness, serial diagnostic tests for Q fever were also per-
formed on the veterinarian (Table 32-2).
Comments: Determination of the date of onset of illness is
essential in any diagnostic investigation of Q fever in people
for correct interpretation of diagnostic test results. In the first
8 to 10 days after the onset of illness, patients are frequently
(80%) bacteremic (and therefore PCR positive) but still
antibody negative. IgM class antibody to phase 2 antigens,
as measured via IFA or ELISA assays, appear as early as 10 to
12 days after the onset of illness, but this may also be
delayed, as occurred in the veterinarian in this example. IgM
class antibody to phase 1 and IgG class antibody to phase
2 antigens often appear on day 15 to 20, but again this
can be delayed. Small animal veterinarians and veterinary
technicians or nurses who are exposed to kittens and placental
secretions and subsequently develop illness should inform
their practitioner of the possibility of exposure to C. burnetii
because diagnosis requires a high index of suspicion and
specific diagnostic assays. Fatigue often persists in patients
for months after the onset of illness (as was the case for
the veterinary technician in this example) and does not
necessarily mean that chronic Q fever has developed.

TABLE 32-2
Results of Diagnostic Assays for Q fever in a Veterinarian and Veterinary Technician after Exposure to an Infected
Cat’s Reproductive Secretions

Real-Time PCR on Whole Serology for IgM and IgG to Phase 1 and Phase 2 Antigens*
Days after Blood
the Onset of
Illness Veterinarian Technician Veterinarian Technician
10 ND Positive ND Negative for IgG and IgM to phase
2 antigen
14 Negative ND Negative ND
20 Negative Negative Negative Positive for IgM to phase 1 and
phase 2 antigens, positive for IgG
to phase 2 antigen. Positive CFT
for antibodies to phase 2 antigen
35 ND ND Positive for IgM and IgG to ND
phase 2 antigen.
Positive CFT for antibodies to
phase 2 antigens

CFT, Complement fixation test; ND, not determined.

*Performed by immunofluorescent antibody unless otherwise specified.

SUGGESTED READINGS
Angelakis E, Raoult D. Q Fever. Vet Microbiol. 2010;140:297-309.
Porter SR, Czaplicki G, Mainil J, et al. Q Fever: current state of knowl-
edge and perspectives of research of a neglected zoonosis. Int J Micro-
biol. 2011;2011:248-418.
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8. Raoult D, Marrie T, Mege J. Natural history and pathophysiology
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14. Seshadri R, Paulsen IT, Eisen JA, et al. Complete genome sequence
of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci U S A.
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15. Angelakis E, Raoult D. Q Fever. Vet Microbiol. 2010;140:297-309.
16. Cairns K, Brewer M, Lappin MR. Prevalence of Coxiella burnetii
DNA in vaginal and uterine samples from healthy cats of north-
central Colorado. J Feline Med Surg. 2007;9:196-201.
17. Komiya T, Sadamasu K, Kang MI, et al. Seroprevalence of Coxiella
burnetii infections among cats in different living environments.
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18. Willeberg P, Ruppanner R, Behymer DE, et  al. Environmental
exposure to Coxiella burnetii: a sero-epidemiologic survey among
domestic animals. Am J Epidemiol. 1980;111:437-443.
19. Skerget M, Wenisch C, Daxboeck F, et al. Cat or dog ownership
and seroprevalence of ehrlichiosis, Q fever, and cat-scratch disease.
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20. Anderson AD, Baker TR, Littrell AC, et al. Seroepidemiologic sur-
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28. Omsland A, Cockrell DC, Howe D, et al. Host cell-free growth of
the Q fever bacterium Coxiella burnetii. Proc Natl Acad Sci U S A.
2009;106:4430-4434.
29. Porter SR, Czaplicki G, Mainil J, et  al. Q Fever: current state of
knowledge and perspectives of research of a neglected zoonosis. Int
J Microbiol. 2011;2011:248-418.
30. La Scola B, Raoult D. Serological cross-reactions between Barton-
ella quintana, Bartonella henselae, and Coxiella burnetii. J Clin
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31. Lukacova M, Melnicakova J, Kazar J. Cross-reactivity between
Coxiella burnetii and chlamydiae. Folia Microbiol (Praha).
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32. Moore JD, Barr BC, Daft BM, et al. Pathology and diagnosis of Cox-
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in placentas from ruminant abortions. APMIS. 2007;115:347-353.
34. Carcopino X, Raoult D, Bretelle F, et al. Managing Q fever during
pregnancy: the benefits of long-term cotrimoxazole therapy. Clin
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35. Astobiza I, Barandika JF, Hurtado A, et  al. Kinetics of Coxiella
burnetii excretion in a commercial dairy sheep flock after treatment
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36. Racult D, Stein A. Q fever during pregnancy—a risk for women,
fetuses, and obstetricians. N Engl J Med. 1994;330:371.
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CHAPTER 33
Chlamydial Infections
Jane E. Sykes
Overview of Chlamydial Infections in Dogs
and Cats
First Described in Cats: 1944, United States (Baker)1
Causes: Chlamydia felis (and possibly other chlamydiae such
as Chlamydia pneumoniae) causes ocular disease in cats;
Chlamydia psittaci and Chlamydia abortus have rarely
been detected in association with ocular and respiratory
disease in dogs. These are obligately intracellular bacteria
that belong to the family Chlamydiaceae.
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact with respiratory secre-
tions and possibly also vaginal secretions and feces
Major Clinical Signs (Cats): Acute and chronic conjunctivi-
tis, chemosis, serous to mucopurulent ocular discharge,
and blepharospasm, with or without sneezing and nasal
discharge
Differential Diagnoses: The primary differential diagnoses in
cats are infections with feline herpesvirus 1, feline calici-
virus, Mycoplasma spp., and/or Bordetella bronchiseptica
Human Health Significance: C. felis has rarely been docu-
mented as a cause of human conjunctivitis. The impor-
tance of cats and dogs as a source of other human
chlamydial infections is unclear. C. psittaci is generally
acquired from avian species and not dogs and cats.
Etiology Agent and Epidemiology
Chlamydiae are obligately intracellular bacteria that belong to
the order Chlamydiales. They primarily cause conjunctivitis in
cats, but may also play a role in other systemic and reproduc-
tive disorders in dogs and cats that remain poorly character-
ized. Most knowledge of chlamydial infections results from
research on human chlamydial pathogens such as Chlamydia
trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci,
which have significant clinical and public health importance (see
Public Health Significance). Chlamydiae structurally resemble
gram-negative bacteria, lack peptidoglycan, and have extremely
small genomes; they depend on the host cell for many amino
acids and nucleotides. They exist in two forms, an infectious
elementary body (EB), which is 0.2 to 0.6 µm in diameter and
exists outside the cell, and the reticulate body (RB), which is
larger (0.5 to 1.5 µm), is noninfectious, and replicates within the
cytoplasm of host cells, before it matures and forms EBs (Figure
33-1). The EBs survive only a few days in the environment at
326
room temperature and they are readily inactivated by most dis-
infectants. Transmission occurs via direct contact or, to a lesser
extent, aerosols. Fomites are also likely to be an important
means of transmission among group-housed cats in heavily con-
taminated environments. The chlamydiae at one time were sepa-
rated into two genera, Chlamydia and Chlamydophila, based
on sequence analysis of the 16S rRNA and 23S rRNA genes.
However, there has been disagreement in the field regarding this
classification, and reversion to the single genus Chlamydia has
since been recommended based on the availability of complete
genome sequence information.2
Chlamydiae persistently infect epithelial cells of the ocular,
respiratory, gastrointestinal, and genitourinary systems; chla-
mydial infections of the synovium have also been reported in
a number of different host species. Different chlamydial species
tend to associate with certain host species, although evidence
has accumulated that some chlamydial species can cause disease
in more than one host species (Table 33-1). In addition, more
than one chlamydial species can cause similar disease manifesta-
tions in a single host species. For example, although the human
ocular disease trachoma is caused primarily by C. trachomatis,
it has now also been associated with infection by C. pneumoniae
and C. psittaci.3 Chlamydia felis is an important cause of con-
junctivitis and possibly reproductive disease in cats, but DNA
that resembles that of the human pathogen C. pneumoniae has
now also been detected in ocular swabs from European cats with
conjunctivitis.4 Chlamydia abortus DNA was detected in histo-
pathology specimens from 1 of 13 cats with arteriosclerosis.5
There are rare reports of chlamydial infections in dogs, in asso-
ciation with ocular and respiratory disease, polyarthritis, and
atherosclerosis.6-11 However, chlamydiae were not detected by
PCR assay in atherosclerotic lesions from 16 dogs in one study,
so the role of chlamydiae in this otherwise rare condition in dogs
is uncertain.5 Respiratory disease associated with C. psittaci
infection has been reported most frequently among dogs.
The most significant pathogen of cats is C. felis, which causes
acute and chronic conjunctivitis in cats worldwide. The results of
genetic analysis (by multilocus variable-number tandem-repeat
and multilocus sequence typing) have revealed that as many as
25 different genotypes of C. felis exist, and more than one geno-
type may circulate in some catteries.12 Co-infections with more
than one strain can occur. The prevalence of infection varies
from one group of cats to another and with geographic location,
age of the cat population sampled, and presence or absence of
clinical illness. Infection occurs most often in ­multiple-cat house-
holds, especially breeding catteries. C. felis DNA is uncommonly
detected in conjunctival swabs from healthy cats. For example,
in clinically healthy household cats, those with a history of past
conjunctivitis, and those with active conjunctivitis, 0%, 4.6%,
 CHAPTER 33  Chlamydial Infections 327

Attachment
Phagosome fusion and 7.3% had positive PCR assay results, respectively.13 Isola-
Reorganization tion rates of up to 30% in household cats with conjunctivitis
and ingestion RB EB to RB
have been reported.14 In shelter cats, the prevalence of C. felis
EB 2hr 8hr
infection has ranged from 0% to 15%.15-18 In cats from 218
8-18hr Multiplication European rescue shelters, breeding establishments, and private
AB of RB households, the prevalence of infection in cats without respi-
ratory disease was 3%, versus 10% in those with evidence of
respiratory disease.19 Infection with C. felis in this European
Persistence
18-24hr study was associated with suboptimal hygiene. Infection with
C. felis is more often present in young cats, especially those aged
Extrusion and 2 to 12 months; kittens less than 2 months of age may be pro-
release of 48-72hr Condensation tected by maternal antibody, although neonatal infections have
infectious EBs 24-48hr RB to EB been described. Cats older than 5 years of age are very unlikely
A Mature inclusion to be infected with C. felis. Co-infections with other respiratory
pathogens, such as feline herpesvirus 1 (FHV-1) and feline cali-
civirus (FCV), are common.
The DNA of organisms that belong to the family Parachla-
mydiaceae has recently been detected in cats with ocular disease.
These organisms resemble Chlamydia and reside symbiotically
within amoebae. Parachlamydial species include ­Neochlamydia,
Parachlamydia, Protochlamydia, ­Rhabdochlamydia, ­Criblamydia,
Simkania, and Waddlia species. DNA that resembles that of
­Neochlamydia hartmanellae has been detected in feline conjunc-
tival brush specimens.20 This organism is an endosymbiont of the
amoeba Hartmannella vermiformis, which has been identified as
B a cause of ocular surface infection in people and may play some
role in feline ocular infections. The DNA of Parachlamydia acan-
FIGURE 33-1  A, Life cycle of chlamydiae. From top left. Electron-dense elementary thamoebae has also been detected with PCR in cats with keratitis
bodies (EBs) attach to and are taken up by epithelial cells. EBs then differentiate into and conjunctivitis.21 As yet, co-infections with amoebae have not
reticulate bodies (RBs), and RBs divide by binary fission. RBs condense into EBs, and also been detected in cats. Further studies are required to determine the
have the potential to persist in the form of aberrant bodies (AB). EBs are released by cell
prevalence and clinical significance of these organisms.
lysis and infect other cells. B, Electron micrograph of chlamydial organisms growing in an
inclusion in tissue culture. The larger reticulate bodies (RBs) have more diffuse chromatin.
One of the RBs appears to be dividing. The smaller dense bodies are elementary bodies.   Clinical Features
(B from Mandell GL, Bennett JE, Dolin R. Principles and Practice of Infectious Diseases. Ed
7. Philadelphia, PA: Elsevier, 2012; 2:2440) Signs and Their Pathogenesis
C. felis infection is acquired by cats primarily through close
contact, fomites, or to a lesser extent, aerosol transmission. Kit-
tens may be infected from the mother at birth. Whether venereal

TABLE 33-1
Chlamydial Species That Infect Animals and Humans
Species Major Host(s)* Major Clinical Manifestations
Chlamydia trachomatis Humans Conjunctivitis (serovars A-C); urethritis, ­cervicitis,
proctitis (serovars D-K); lymphogranuloma
­venereum (serovars L1, L2, and L3)
Chlamydia pneumoniae Humans Upper respiratory infection, community-acquired
pneumonia, possibly ­atherosclerosis and asthma
Chlamydia psittaci Humans, a variety of bird species (e.g., parrots, Pneumonia, systemic ­infection
finches, poultry, pigeons, pheasants, seagulls,
egrets, puffins)
Chlamydia felis Cats Conjunctivitis, upper respiratory infection, arthritis?,
reproductive disease?
Chlamydia abortus Sheep, cows, goats, other mammals Reproductive disease
Chlamydia pecorum Cattle and sheep Encephalomyelitis, pneumonia, reproductive tract
disease, conjunctivitis, polyarthritis
Chlamydia caviae Guinea pigs Keratoconjunctivitis

*Infection may occasionally extend to other host species.

328 SECTION 2  Bacterial Diseases
transmission occurs, as reported for other chlamydial species,
is unknown, but in some infected cats, the organism is shed
in vaginal discharges and from the rectum as well as in ocu-
lar discharges. After uptake of EBs, the EB transitions into the
RB, which replicates by binary fission in a membrane-enclosed
vacuole called an inclusion and avoids lysosomal fusion. The
RBs then transition back to the EB form, which is released into
the extracellular milieu after cell lysis occurs and subsequently
infect other host cells. The entire replication cycle takes about
2 days to complete (see Figure 33-1).22
Chlamydiae primarily infect epithelial cells, but they can
also infect a variety of other cell types including endothelial
cells, smooth muscle cells, lymphocytes, monocytes, and mac-
rophages. C. felis replicates in the cytoplasm of conjunctival
epithelial cells, but also spreads via the bloodstream to a variety
of other tissues, such as the tonsil, lung, liver, spleen, gastroin-
testinal tract, and kidney.23 Infection is typically followed 2 to
5 days later by the development of acute, chronic, or recurrent
conjunctivitis, with or without signs of rhinitis such as nasal dis-
charge and sneezing. Most cats remain otherwise well and appe-
tent. Lower respiratory tract signs such as cough and dyspnea
rarely, if ever, occur. Corneal disease such as keratitis or corneal
ulceration is rare and, if present, is likely caused by other or
co-infecting pathogens such as FHV-1. Experimental inocula-
tions have also been followed by signs of fever, lethargy, lame-
ness and joint pain (possibly due to polyarthritis), and weight
loss, in addition to conjunctivitis.24 There are also rare reports
of gastritis and peritonitis in association with the detection of
chlamydia-like organisms in cats.25,26
After the initial episode of acute conjunctivitis, infection may
persist for many months. This may be accompanied by mild
conjunctivitis or, in some cases, no clinical signs of disease.
Organisms can be isolated from the conjunctiva for up to 215
days after experimental infection.27,28 Whether chronic disease
is the result of repeated reinfection or the presence of persis-
tent chlamydiae is unclear. Co-infections with other agents such
as FCV, FHV-1, Bordetella, or Mycoplasma may increase the
severity of disease and duration of shedding. Other bacteria,
including those that normally colonize the healthy conjunctival
sac, also act as secondary invaders and worsen disease.
Whether C. felis causes reproductive disease in cats remains
unclear. The stage of pregnancy at which infection occurs,
co-infection with other organisms such as FHV-1, concurrent
immunosuppression, route of infection, and the strain involved
may be important in determining the ability of C. felis to cause
reproductive dysfunction, if this indeed occurs in cats.
Chlamydial infections are infrequently described in dogs.
Chlamydia psittaci has been detected in dogs with respiratory
disease, such as pneumonia and pleural effusion, and/or kera-
toconjunctivitis.6-8 One group of bitches that had ocular and
lower respiratory signs as well as stillbirths and low litter size
had nasal, conjunctival, and pharyngeal swab specimens that
tested positive for C. psittaci with a PCR assay.6 Chlamydia
abortus was detected in a dog with keratoconjunctivitis.9 The
DNA of C. pneumoniae was detected in atherosclerotic lesions
from dogs in Japan,10 and chlamydia-like organisms were found
in a dog from South Africa with polyarthritis.11
Physical Examination Findings
Physical examination findings in cats with chlamydiosis
include conjunctivitis, chemosis, serous to mucopurulent ocu-
lar discharge, and blepharospasm (Figure 33-2). Signs of nasal
FIGURE 33-2  Domestic shorthair cat with acute chlamydial conjunctivitis.
Marked hyperemia and chemosis are present, as well as serous to mucopurulent ocular
discharge.
involvement, such as stertorous respiration, serous or mucopu-
rulent nasal discharge, and sneezing, may accompany conjuncti-
vitis. Mucopurulent vaginal discharge has been reported in some
cats that were experimentally infected by topical ophthalmic
inoculation.
Physical examination findings described in dogs with chla-
mydiosis have included fever, tachypnea, keratoconjunctivitis
with mucopurulent ocular discharge, shifting leg lameness, and
lymphadenopathy.6-9,11
Diagnosis
There are no clinically significant laboratory abnormalities in
cats with chlamydiosis, and there is insufficient information
on laboratory abnormalities that might occur in affected dogs.
Diagnosis of chlamydiosis is most often based on the results of
molecular testing using PCR assays (Table 33-2). For all diag-
nostic tests that detect chlamydial organisms, sufficient num-
bers of infected epithelial cells must be collected. This generally
involves vigorous swabbing of infected mucosal sites such as the
conjunctiva.
Microbiologic Tests
Cytologic Examination for Inclusions
Using light microscopy, chlamydial intracytoplasmic inclusions
may be seen in epithelial cells that are present in smears of con-
junctival scrapings. Inclusions are made up of basophilic clus-
ters of coccoid bacteria and are up to 10 µm in diameter (Figure
33-3). Giemsa is the preferred staining method. Inclusions are
only generally visible early, if at all, during the course of infec-
tion, so this method is insensitive. Also, melanin granules in the
cytoplasm of conjunctival epithelial cells can yield false-positive
results. The use of direct fluorescent antibody staining or immu-
nocytochemical stains that detect Chlamydia increases sensitiv-
ity and specificity.
Enzyme-Linked Immunosorbent Assay Antigen Detection
A variety of commercial antigen detection enzyme immunoassay
kits have been developed and marketed for diagnosis of human
chlamydial infections. Nucleic acid amplification tests have now
 CHAPTER 33  Chlamydial Infections 329

TABLE 33-2
Diagnostic Assays Available for Chlamydiosis in Dogs and Cats
Assay Specimen Type Target Performance
Cell culture Conjunctival swabs Chlamydia spp. Not widely offered or utilized for routine diagnostic pur-
poses. Requires special chlamydial transport media. False
negatives occur when organisms lose viability during
transport or in chronic infections, when organism numbers
are very low.
Cytology Conjunctival smears Chlamydial inclusions Low sensitivity. Inclusions are most likely to be seen early
in infection. False positives can occur when cytoplasmic
granules are mistaken for inclusions. Fluorescent or immu-
noperoxidase antibody techniques may increase sensitivity
and specificity.
Serology Serum Antibodies to Acute and convalescent serology is required for diagnosis of
Chlamydia spp. acute infection. Vaccination may interfere with interpreta-
tion of results. Not widely available or utilized worldwide.
PCR Conjunctival swabs, Chlamydia felis DNA or Confirms active infection. Assays may be genus or species
scrapings, or biopsies Chlamydia spp. DNA specific. Sensitivity and specificity may vary depending on
assay design and specimen type. Because healthy animals
occasionally test PCR positive, positive PCR results must
be interpreted in light of the clinical signs. Vaccination with
attenuated live chlamydial vaccines has the potential to
interfere with results.

antigen have lower sensitivity and specificity than cell culture,

direct fluorescent antibody methods, or PCR assays for detec-
tion of C. felis.

FIGURE 33-3  Conjunctival scraping from a cat with chlamydial conjunctivitis.
Inclusions of Chlamydia felis are found in an epithelial cell (arrows). Wright’s stain, original
magnification 1000×. (Courtesy of Judith Taylor, LabVet Consultations, Inc., Guelph, Ontario,
Canada and Karen M. Young, University of  Wisconsin-Madison. In Young KM, Taylor JJ: Labora-
tory medicine: yesterday today tomorrow: eye on the cytoplasm, Vet Clin Pathol 35:141, 2006.)
largely replaced the use of these assays for diagnosis of human
infections. Antibodies used in ELISA assays that are designed to
detect human chlamydial pathogens in swab specimens (such as
conjunctival swabs) cross-react with C. felis antigen and so have
also been used for diagnosis of feline chlamydiosis. Unfortu-
nately, the kits have varied considerably in their sensitivity and
specificity. In general, ELISA methods that detect chlamydial
Cell Culture
Cell culture has traditionally been considered the gold standard
for chlamydial diagnosis, but is now mostly performed on a
research basis. Isolations from cats are usually highest from con-
junctival swabs, although nasal and pharyngeal swab specimens
may also contain infectious organisms. To enhance organism
survival, swabs should be placed immediately in a chlamydial
transport medium such as 2SP (0.2 M sucrose and 0.02 M phos-
phate), which can be obtained from laboratories that perform
chlamydial culture. Routine viral transport media should not be
used, because it contains antibiotics that inactivate the organisms.
The specimen should be refrigerated (4°C) if it is not immediately
sent to the laboratory and ideally should reach the laboratory
within 24 hours of specimen collection. In the laboratory, organ-
isms are readily grown in the yolk sacs of embryonating eggs or
in cell lines such as McCoy or HeLa cells over a period of 2 to
3 days. Fluorescent antibody is then used to identify chlamydial
inclusions within the infected cell culture (Figure 33-4).
Molecular Diagnosis Using the Polymerase Chain Reaction
The use of PCR-based assays has revolutionized diagnosis of
chlamydial infections in animals and humans, because these
assays are rapid, sensitive, and specific and do not require spe-
cial transport conditions. Sequence analysis of PCR products
can be used to differentiate between species of Chlamydiaceae.
Both conventional and real-time PCR assays have been used to
detect C. felis in conjunctival swab specimens from cats,29-31 and
real-time PCR assays that detect C. felis are now offered by sev-
eral commercial veterinary diagnostic laboratories worldwide.
330 SECTION 2  Bacterial Diseases
FIGURE 33-4  Chlamydial inclusions stained with fluorescent antibody in a mccoy cell
monolayer. (Courtesy of Robert Suchland, Seattle, In Stamm WE, Batteiger BE. Introduc-
tion to Chlamydia and Chlamydophila. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell,
Douglas and Bennett’s Principles and Practice of Infectious Diseases, 7 ed. Philadelphia,
PA: Churchill Livingstone Elsevier; 2011.)
Unfortunately, considerable interlaboratory variation in assay
performance still exists.32 In addition, PCR assays for C. felis
may not detect other chlamydial species, such as C. pneumoniae.
Genus- or family-specific chlamydial PCR assays must be used
to detect these organisms. The laboratory should be consulted
to determine the specificity of the assay used. The use of a DNA
microarray assay for detection and identification of chlamydiae
in cats has also been reported.5 The microarray assay consists of
a chip that carries chlamydial species- and genus-specific probes.
DNA within the specimen is first amplified and biotin labeled
with PCR, and then reacted with the microarray probes.33
PCR assays for C. felis are usually more sensitive compared
with cell culture for diagnosis of infection in naturally infected
cats (see Table 33-1).29 During antimicrobial treatment of
infected cats, PCR assay results continue to be positive for sev-
eral days.29,30 Topical ophthalmic solutions such as proxymeta-
caine and fluorescein did not interfere with the ability of
real-time PCR to detect C. felis, but there was a slight inhibitory
effect when fusidic acid was used.34 Because cats without signs
of conjunctivitis occasionally test PCR positive for C. felis, the
results of PCR must be interpreted in light of the history, clinical
signs present, and response to appropriate treatment.
Serologic Testing
Serologic tests are not widely available for routine diagnosis
of chlamydial infections. Assays that detect serum antibod-
ies to chlamydiae, such as IFA or ELISA assays, are of limited
benefit for detection of active infection because the IgG anti-
body titer increase is variable or prolonged, and IgM antibody
titers are inconsistently increased. However, very high anti-
body titers (≥1:512) have been associated with clinical illness
and/or chlamydial shedding, and high antibody titers among
group-housed cats with conjunctivitis can suggest a role for
C. felis in disease.13,23,35 Chlamydial infections persist in the
presence of high serum antibody titers, so positive antibody
titers after vaccination do not imply protection. Serial titers may
be needed to document active infections, and recent vaccination
may interfere with interpretation of positive titer results. An
ELISA assay that discriminates between the antibody response
from vaccination and that from natural infection has been
described but is not widely available on a commercial basis.36
TABLE 33-3
Suggested Medications for Treatment of Cats
with Ocular Chlamydiosis
Interval
Drug Dose Route (hours) Duration
Doxycycline* 10 mg/kg PO 24 At least 4 weeks,
or 2 weeks after
resolution of
­clinical signs
Amoxicillin- 12.5 PO 12 4 weeks
clavulanate
Pradofloxacin 5-7.5† PO 24 6 weeks
*Doxycycline is the treatment of choice. For cats that fail to tolerate
doxycycline, amoxicillin-clavulanate should be used. Fluoroquinolones
are an option for cats that do not tolerate doxycycline or amoxicillin-
clavulanate.
†Dose is for the oral suspension; if tablets are used, the dose is lower
(3 mg/kg).
Treatment and Prognosis
Chlamydial infections are generally treated with tetracycline
antibiotics. Oral administration of doxycycline at 5 to 10
mg/kg q12h for 3 to 4 weeks results in clinical resolution in
most cats. A dose of 10 mg/kg q24h can also be used (Table
33-3).23 Clinical improvement occurs within 24 to 48 hours
after treatment is initiated. In colonies of cats, all cats should
be treated for 4 weeks: in one study that used real-time PCR
to detect infection, a 3-week course of treatment was insuf-
ficient to clear infection in some cats, but treatment for 28
days subsequently cleared infection in all cats.30 Treatment
for up to 6 to 8 weeks may be required. Regardless, treatment
should continue for at least 2 weeks after the resolution of
clinical signs.
Failure to respond completely to treatment may reflect the
concurrent presence of viral upper respiratory tract pathogens.
Because administration of doxycycline hyclate can result in
esophagitis and esophageal stricture formation, the administra-
tion of doxycycline hyclate tablets should be followed by a bolus
of water or food (see Chapter 8 for additional information on
doxycycline). Ocular infections may initially respond to tetracy-
cline eye ointment applied two or three times daily,37 but recur-
rent infections have been documented,38 likely because systemic
infection with chlamydiae can occur in cats with conjunctivitis.
For cats that do not tolerate doxycycline, 4 weeks of
amoxicillin-clavulanate is a suitable alternative.39 In exper-
imentally infected cats, the efficacy of enrofloxacin (5 mg/
kg PO q24h for 14 days) was similar to that of doxycycline
as determined with direct fluorescent antibody on conjunc-
tival swabs, although a few cats in both the doxycycline and
enrofloxacin groups had evidence of persistent infection at
the end of the treatment period.40 Doxycycline is preferred
because of concerns that relate to the possibility of reti-
nal toxicity in cats treated with enrofloxacin. Doxycycline
was more efficacious than pradofloxacin in experimentally
infected cats that were monitored with a PCR assay.41 Sul-
fonamides or chloramphenicol are ineffective, and in  vitro,
penicillin is only inhibitory at higher doses. Treatment with

azithromycin is also incompletely effective when compared
with doxycycline.42
Immunity and Vaccination
Kittens acquire maternal antibodies to C. felis that usually
protect them from infection until 7 to 9 weeks of age. Both
humoral and cell-mediated mechanisms provide protective
immunity in chlamydial infections of other host species. Nat-
ural infection seems to confer little protection against reinfec-
tion, and this protection appears to be short-lived. However,
given the low prevalence of chlamydiosis in older cats, an
age-related resistance to chlamydiosis appears to exist.
Both inactivated and attenuated live vaccines are available
to protect cats from chlamydial respiratory disease. These
vaccines do not entirely prevent infection and shedding of
chlamydiae after challenge, but they minimize the replica-
tion of the organism and hence reduce the severity of clini-
cal signs. Transient fever, anorexia, lethargy, and lameness
has been described 1 to 3 weeks after use of combined vac-
cines containing attenuated live C. felis in a low percentage
of cats. Chlamydial vaccines are noncore vaccines that are
recommended for cats with a high risk of acquiring infec-
tion, such as those introduced to or that reside in catteries
with documented endemic chlamydiosis. These cats should
be vaccinated regularly after initial treatment of all cats with
appropriate antimicrobial drugs.
Prevention
Given that chlamydiae survive poorly in the environment and
the major route of transmission is close contact, transmission
can be minimized by good hygiene, quarantine, single housing,
and disinfection practices in cattery situations. Chlamydiae are
readily inactivated by detergent solutions.
CASE EXAMPLE
Signalment: “Sam,” a 3-year-old male neutered domestic
shorthair cat from Davis, CA
History: Sam was brought to the University of California,
Davis, by a veterinary student for evaluation of fever,
right thoracic limb lameness, and bilateral conjunctivitis
with mucopurulent ocular discharge of 2 days’ duration.
No coughing, sneezing, vomiting, diarrhea, or abnormal
thirst or urination had been noted. Sam lived indoors with
5 other cats, 3 of which had also had severe conjunctivitis,
ocular discharge, fever, lethargy, and lameness for the
last week. These 3 cats ranged from 8 months to 2.5
years of age. All of the cats had been vaccinated regularly
for FHV-1, FCV, and feline panleukopenia virus and
tested retrovirus-negative as kittens. They were all fed a
commercial dry cat food.
Physical Examination:
Body Weight: 6.7 kg
General: Bright, alert and responsive, hydrated. T = 103.6°F
(39.7°C), P = 240 beats/min, respiratory rate 66 breaths/min,
mucous membranes pink, CRT = 1 s.
CHAPTER 33  Chlamydial Infections 331
Public Health Aspects
The most significant chlamydial pathogens of humans are C.
trachomatis, C. pneumoniae, and C. psittaci. C. trachoma-
tis causes trachoma, the leading cause of infectious blindness
worldwide, and it also is the most common bacterial cause of
sexually transmitted diseases in humans.22 C. pneumoniae may
infect nearly all humans, and is an important cause of commu-
nity-acquired pneumonia worldwide. It has also been impli-
cated in atherosclerotic cardiovascular disease, although its role
in this condition remains unclear. C. psittaci causes psittacosis
in birds and humans, a potentially life-threatening zoonosis.
C. felis has long been suspected as a cause of conjunc-
tivitis in humans. However, many of the reports of human
infection with C. felis before the advent of genetic detection
methods remain unconfirmed, because diagnosis was based
either on culture, without identification to the species level,
or on serologic testing, which has the potential to cross-react
with other chlamydiae and organisms such as Bartonella.
More recently, molecular assays have been used to confirm
C. felis infection in humans. C. felis was detected in the con-
junctiva of a person with conjunctivitis.24 The organism was
genetically indistinguishable from an isolate later recovered
from the patient’s cat. Although these associations have been
difficult to document, routine precautions should be taken
when handling cats with conjunctivitis, especially young cats
with chronic conjunctivitis. Proper hand washing and routine
cleaning and disinfection are likely to be sufficient to prevent
transmission.
The identification of C. psittaci in dogs with respiratory dis-
ease and C. pneumoniae in cats with ocular disease suggests
that dogs and cats may have the potential to be a source of
these pathogens for humans, although avian species and other
infected people, respectively, are likely to be much more signifi-
cant sources of infection.
Eyes, Ears, Nose, and Throat: There was no evidence of ocular
discharge, but mild conjunctival hyperemia was present.
The nasal planum was clean and moist with no discharge;
no oral erosions or ulcerations were seen.
Musculoskeletal: Body condition score 7/9. Decreased weight
bearing on the left thoracic limb was noted, and the cat
sometimes held his left thoracic limb up when sitting. There
was possible mild effusion in the left elbow and resentment
on flexion of the left carpus and both elbows.
All Other Systems: No clinically significant abnormalities were
detected.
Laboratory Findings:
CBC:
HCT 46.3% (30%-50%)
MCV 46.9 fL (42-53 fL)
MCHC 30.7 g/dL (30-33.5 g/dL)
WBC 10,500 cells/µL (4500-14,000 cells/µL)
Neutrophils 7455 cells/µL (2000-9000 cells/µL)
Lymphocytes 2310 cells/µL (1000-7000 cells/µL)
Monocytes 525 cells/µL (50-600 cells/µL)
Eosinophils 210 cells/µL (150-1100 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL)
Platelets 174,000/µL (180,000-500,000 platelets/µL), with
­moderate macroplatelets.
332 SECTION 2  Bacterial Diseases
Serum Chemistry Profile:
Sodium 152 mmol/L (151-158 mmol/L)
Potassium 4.4 mmol/L (3.6-4.9 mmol/L)
Chloride 117 mmol/L (117-126 mmol/L)
Bicarbonate 18 mmol/L (15-21 mmol/L)
Phosphorus 4.0 mg/dL (3.2-6.3 mg/dL)
Calcium 9.5 mg/dL (9.0-10.9 mg/dl)
BUN 21 mg/dL (18-33 mg/dL)
Creatinine 1.6 mg/dL (1.1-2.2 mg/dL)
Glucose 151 mg/dL (63-118 mg/dL)
Total protein 7.1 g/dL (6.6-8.4 g/dL)
Albumin 3.1 g/dL (2.2-4.6 g/dL)
Globulin 4.0 g/dL (2.8-5.4 g/dL)
ALT 48 U/L (27-101 U/L)
AST 8 U/L (17-58 U/L)
ALP 31 U/L (14-71 U/L)
Gamma GT 0 U/L (0-4 U/L)
Cholesterol 154 mg/dL (89-258 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.055; pH 6.5, trace protein, no hemoprotein,
glucose, ketones or bilirubin, 0-1 WBC/HPF, 0-2 RBC/HPF, few
lipid droplets.
Synovial Fluid Cytology: Synovial fluid from the right stifle,
right carpus, left carpus, and right tarsus was examined.
All specimens apart from that from the left carpus were
markedly blood contaminated and so could not be
properly evaluated. The cell counts in those specimens
ranged from 2000 to 4000 nucleated cells/µL and consisted
predominantly of neutrophils (66%-80%) and small
mononuclear cells (7%-18%). The specimen from the left
carpus had 2000 nucleated cells/µL, of which 14% were
neutrophils, 85% large mononuclear cells, and 1% small
mononuclear cells. There were moderate numbers of
erythrocytes and blood-associated leukocytes, as well
as low numbers of large mononuclear cells of normal
morphology, which was consistent with mild blood
contamination.
SUGGESTED READINGS
Gruffydd-Jones T, Addie D, Belak S, et al. Chlamydophila felis infec-
tion. ABCD guidelines on prevention and management. J Feline Med
Surg. 2009;11:605-609.
Laroucau K, Di Francesco A, Vorimore F, et  al. Multilocus variable-
number tandem-repeat analysis scheme for Chlamydia felis genotyp-
ing: comparison with multilocus sequence typing. J Clin Microbiol.
2012;50(6):1860-1866.
Sibitz C, Rudnay EC, Wabnegger L, et  al. Detection of Chla-
mydophila pneumoniae in cats with conjunctivitis. Vet Ophthalmol.
2011;14(suppl 1):67-74.
REFERENCES
1. Baker JA. A virus causing pneumonia in cats and producing ele-
mentary bodies. J Exp Med. 1944;79(2):159-172.
2. Stephens RS, Myers G, Eppinger M, et  al. Divergence without
difference: phylogenetics and taxonomy of Chlamydia resolved.
FEMS Immunol Med Microbiol. 2009;55:115-119.
3. Dean D, Kandel RP, Adhikari HK, et al. Multiple Chlamydiaceae
species in trachoma: implications for disease pathogenesis and con-
trol. PLoS Med. 2008;5:e14.
Conjunctival Smear Cytology: Chlamydial inclusions were
visualized within several epithelial cells.
Imaging Findings: Radiographs of the left radius and
ulna, including carpus and elbow joints: no radiographic
abnormalities.
Microbiologic Testing: FeLV antigen and FIV antibody
serology: negative
PCR for FCV (oropharyngeal swab specimens): negative
Mycoplasma culture (conjunctival swab specimen): negative
Diagnosis: A tentative diagnosis of chlamydial conjunctivitis
and arthritis was made.
Treatment and Outcome: Treatment of all cats in the
household with doxycycline (10 mg/kg PO q24h) was
initiated. This was associated with rapid resolution of fever,
ocular signs, and lameness.
Comments: The cats in this household had conjunctivitis and
possibly arthritis in association with chlamydial infection.
The major differential diagnosis was FCV infection, which
is more widespread than chlamydiosis and for which an
association between lameness and infection has been
described. Infection with Mycoplasma spp. has also
been associated with conjunctivitis as well as synovitis
in cats (see Chapter 40). Lameness has developed in cats
that were experimentally infected with C. felis, but there
are no reports of lameness in association with natural
infection. The negative PCR and culture results for FCV
and Mycoplasma spp., respectively, could not definitively
rule out infection with these agents. The presence of
chlamydial inclusions in the conjunctival smear supported
a diagnosis of chlamydiosis, but false positives can occur,
and unfortunately PCR was not performed to confirm
the presence of a chlamydial agent. Nevertheless, the
rapid response to doxycycline supported the possibility
of a bacterial cause of the arthritis. Interestingly,
thrombocytopenia was present (and confirmed on smear
evaluation), which has been reported in humans with
chlamydial infections.43
4. Sibitz C, Rudnay EC, Wabnegger L, et  al. Detection of Chla-
mydophila pneumoniae in cats with conjunctivitis. Vet Ophthal-
mol. 2011;14(suppl 1):67-74.
5. Sostaric-Zuckermann IC, Borel N, Kaiser C, et al. Chlamydia in canine
or feline coronary arteriosclerotic lesions. BMC Res Notes. 2011;4:350.
6. Sprague LD, Schubert E, Hotzel H, et  al. The detection of
Chlamydophila psittaci genotype C infection in dogs. Vet J.
2009;181:274-279.
7. Gresham AC, Dixon CE, Bevan BJ. Domiciliary outbreak of psittacosis
in dogs: potential for zoonotic infection. Vet Rec. 1996;138:622-623.
8. Arizmendi F, Grimes JE, Relford RL. Isolation of Chlamydia psittaci
from pleural effusion in a dog. J Vet Diagn Invest. 1992;4:460-463.
9. Hoelzle K, Wittenbrink MM, Corboz L, et al. Chlamydophila abortus-
induced keratoconjunctivitis in a dog. Vet Rec. 2005;157:632-633.
10. Sako T, Takahashi T, Takehana K, et al. Chlamydial infection in
canine atherosclerotic lesions. Atherosclerosis. 2002;162:253-259.
11. Lambrechts N, Picard J, Tustin RC. Chlamydia-induced septic
polyarthritis in a dog. J S Afr Vet Assoc. 1999;70:40-42.
12. Laroucau K, Di Francesco A, Vorimore F, et  al. Multilocus
­variable-number tandem-repeat analysis scheme for Chlamydia
felis genotyping: comparison with multilocus sequence typing.
J Clin Microbiol. 2012;50(6):1860-1866.

13. Low HC, Powell CC, Veir JK, et al. Prevalence of feline herpesvirus
1, Chlamydophila felis, and Mycoplasma spp. DNA in conjunctival
cells collected from cats with and without conjunctivitis. Am J Vet
Res. 2007;68:643-648.
14. Wills JM, Howard PE, Gruffydd-Jones TJ, et  al. Prevalence of
Chlamydia psittaci in different cat populations in Britain. J Small
Anim Pract. 1988;29:327-339.
15. Kang BT, Park HM. Prevalence of feline herpesvirus 1, feline calici-
virus and Chlamydophila felis in clinically normal cats at a Korean
animal shelter. J Vet Sci. 2008;9:207-209.
16. Veir JK, Ruch-Gallie R, Spindel ME, et al. Prevalence of selected
infectious organisms and comparison of two anatomic sampling
sites in shelter cats with upper respiratory tract disease. J Feline
Med Surg. 2008;10:551-557.
17. Maggs DJ, Sykes JE, Clarke HE, et  al. Effects of dietary lysine
supplementation in cats with enzootic upper respiratory disease.
J Feline Med Surg. 2007;9:97-108.
18. Bannasch MJ, Foley JE. Epidemiologic evaluation of multiple
respiratory pathogens in cats in animal shelters. J Feline Med Surg.
2005;7:109-119.
19. Helps CR, Lait P, Damhuis A, et  al. Factors associated with
upper respiratory tract disease caused by feline herpesvirus,
feline calicivirus, Chlamydophila felis and Bordetella bronchi-
septica in cats: experience from 218 European catteries. Vet Rec.
2005;156:669-673.
20. von Bomhard W, Polkinghorne A, Lu ZH, et  al. Detection of
novel chlamydiae in cats with ocular disease. Am J Vet Res.
2003;64:1421-1428.
21. Richter M, Matheis F, Gonczi E, et al. Parachlamydia acantham-
oebae in domestic cats with and without corneal disease. Vet Oph-
thalmol. 2010;13:235-237.
22. Saka HA, Thompson JW, Chen YS, et al. Quantitative proteomics
reveals metabolic and pathogenic properties of Chlamydia tracho-
matis developmental forms. Mol Microbiol. 2011;82:1185-1203.
23. Gruffydd-Jones T, Addie D, Belak S, et  al. Chlamydophila felis
infection. ABCD guidelines on prevention and management.
J Feline Med Surg. 2009;11:605-609.
24. TerWee J, Sabara M, Kokjohn K, et  al. Characterization of the
systemic disease and ocular signs induced by experimental infection
with Chlamydia psittaci in cats. Vet Microbiol. 1998;59:259-281.
25. Dickie CW, Sniff ES. Chlamydia infection associated with peritoni-
tis in a cat. J Am Vet Med Assoc. 1980;176:1256-1259.
26. Hargis AM, Prieur DJ, Gaillard ET. Chlamydial infection of the
gastric mucosa in twelve cats. Vet Pathol. 1983;20:170-178.
27. Wills JM. Chlamydial infection in the cat. University of Bristol:
PhD Thesis; 1986.
28. O’Dair HA, Hopper CD, Gruffydd-Jones TJ, et al. Clinical aspects
of Chlamydia psittaci infection in cats infected with feline immuno-
deficiency virus. Vet Rec. 1994;134:365-368.
29. Sykes JE, Studdert VP, Browning GF. Comparison of the poly-
merase chain reaction and culture for the detection of feline Chla-
mydia psittaci in untreated and doxycycline-treated experimentally
infected cats. J Vet Intern Med. 1999;13:146-152.
CHAPTER 33  Chlamydial Infections 333
30. Dean R, Harley R, Helps C, et  al. Use of quantitative real-time
PCR to monitor the response of Chlamydophila felis infection to
doxycycline treatment. J Clin Microbiol. 2005;43:1858-1864.
31. Helps C, Reeves N, Tasker S, et  al. Use of real-time quantitative
PCR to detect Chlamydophila felis infection. J Clin Microbiol.
2001;39:2675-2676.
32. Sandmeyer LS, Waldner CL, Bauer BS, et al. Comparison of poly-
merase chain reaction tests for diagnosis of feline herpesvirus,
Chlamydophila felis, and Mycoplasma spp. infection in cats with
ocular disease in Canada. Can Vet J. 2010;51:629-633.
33. Borel N, Kempf E, Hotzel H, et  al. Direct identification of chla-
mydiae from clinical samples using a DNA microarray assay: a vali-
dation study. Mol Cell Probes. 2008;22:55-64.
34. Segarra S, Papasouliotis K, Helps C. The in  vitro effects of
proxymetacaine, fluorescein, and fusidic acid on real-time
PCR assays used for the diagnosis of feline herpesvirus 1 and
­Chlamydophila felis infections. Vet Ophthalmol. 2011;14
(suppl 1):5-8.
35. Strom Holst B, Krook L, Englund S, et al. Shedding of chlamydiae
in relation to titers of serum chlamydiae-specific antibodies and
serum concentrations of two acute-phase proteins in cats without
conjunctivitis. Am J Vet Res. 2011;72:806-812.
36. Ohya K, Okuda H, Maeda S, et al. Using CF0218-ELISA to distin-
guish Chlamydophila felis-infected cats from vaccinated and unin-
fected domestic cats. Vet Microbiol. 2010;146:366-370.
37. Sparkes AH, Caney SM, Sturgess CP, et  al. The clinical efficacy
of topical and systemic therapy for the treatment of feline ocular
chlamydiosis. J Feline Med Surg. 1999;1:31-35.
38. Donati M, Piva S, Di Francesco A, et al. Feline ocular chlamydiosis:
clinical and microbiological effects of topical and systemic therapy.
New Microbiol. 2005;28:369-372.
39. Sturgess CP, Gruffydd-Jones TJ, Harbour DA, et  al. Controlled
study of the efficacy of clavulanic acid-potentiated amoxicil-
lin in the treatment of Chlamydia psittaci in cats. Vet Rec.
2001;149:73-76.
40. Gerhardt N, Schulz BS, Werckenthin C, et  al. Pharmacokinetics
of enrofloxacin and its efficacy in comparison with doxycycline in the
treatment of Chlamydophila felis infection in cats with conjunctivitis.
Vet Rec. 2006;159:591-594.
41. Hartmann AD, Helps CR, Lappin MR, et  al. Efficacy of prado-
floxacin in cats with feline upper respiratory tract disease due to
Chlamydophila felis or Mycoplasma infections. J Vet Intern Med.
2008;22:44-52.
42. Owen WM, Sturgess CP, Harbour DA, et al. Efficacy of azithro-
mycin for the treatment of feline chlamydophilosis. J Feline Med Surg.
2003;5:305-311.
43. Day CJE, Fawcett IW. Psittacosis and acute thrombocytopenic pur-
pura. J R Soc Med. 1992;85(6):360-361.
CHAPTER 34

Streptococcal and Enterococcal Infections

Jane E. Sykes

Overview of Streptococcal and Enterococcal
Infections
First Described: Streptococci were first described in Germany
in 1868 by Billroth.1 Enterococci were separated from the
genus Streptococcus in 1984.2
Major Causes: In dogs and cats, the most common causes
are Streptococcus canis, Streptococcus equi subsp. zooepi-
demicus, Enterococcus faecium, and Enterococcus faecalis,
which are gram-positive coccoid bacteria
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact. Organisms are usually
commensals that invade opportunistically.
Major Clinical Signs: Neonatal septicemia, pharyngitis, cervi-
cal lymphadenitis, bacteremia and endocarditis, urinary
tract infections, postoperative incision or wound infec-
tions, otitis externa, keratitis, bronchopneumonia, pyome-
tra or metritis, meningoencephalitis, necrotizing fasciitis
and toxic shock syndrome (primarily S. canis), rhinitis and
necrotizing sinusitis, pyothorax, discospondylitis, arthritis,
osteomyelitis, mastitis, cholangiohepatitis, and peritonitis
Differential Diagnoses: Primarily other gram-positive and gram-
negative bacterial infections
Human Health Significance: Streptococcal species that infect
dogs and cats occasionally infect human patients. Dogs
and cats may also have the potential to serve as sources of
multidrug-resistant enterococcal infections for humans.
humans, streptococci that infect dogs and cats range from com-
mensals of low virulence through to highly virulent organisms
that can cause severe disease manifestations and death.
Streptococci have been classified based on their hemolytic
properties into β-hemolytic streptococci, which cause com-
plete hemolysis when they grow on blood agar; γ-hemolytic or
nonhemolytic streptococci, which do not cause hemolysis; and
α-hemolytic streptococci, which reduce hemoglobin, causing a
greenish discoloration of the agar around bacterial colonies.
Streptococci are also classified from A through W based on their
surface antigens (Lancefield classification system) and classi-
fied based on phenotypic characteristics (such as pyogenic or
viridans group streptococci). Major pathogenic streptococci of
dogs and cats belong to Lancefield groups B, C, D, or G (Table
34-1). Pyogenic streptococci are β-hemolytic streptococci that
belong to Lancefield groups A (S. pyogenes), B (Streptococcus
agalactiae), C (which includes Streptococcus dysgalactiae and
S. equi subsp. zooepidemicus), and G (which includes Strepto-
coccus canis). Viridans streptococci are often nonhemolytic or
α-hemolytic (viridis = Latin for green) and tend to be commen-
sals that have low virulence and rarely invade tissues opportu-
nistically. Many group D streptococci have now been classified
as enterococci. Molecular typing schemes have revealed that
hemolytic reactions, Lancefield antigens, and phenotypic char-
acteristics do not necessarily predict genetic relatedness among

Etiology, Epidemiology, and Clinical Features

Streptococci are catalase-negative, gram-positive cocci that
divide along a single axis, forming pairs and chains of organ-
isms (Figures 34-1 and 34-2). They are facultative to strict
anaerobes that require complex media for growth, preferably
supplemented with blood. More than 40 species of streptococci
have been described. Species of streptococci vary in host tro-
pism and virulence properties, and so it is important that they
be discussed separately. One of the most important streptococ-
cal pathogens of humans, Streptococcus pyogenes, which causes
streptococcal pharyngitis (“strep throat”), does not infect dogs FIGURE 34-1  Histopathology of the serosal surface of the liver of a 12-day-old
and cats to a significant degree (see Public Health Aspects). domestic shorthair kitten that was euthanized after it was found hypothermic and
However, interspecies transmission has been reported for some tachypneic. Severe suppurative omphalophlebitis and peritonitis was present. Lesions
pathogenic streptococci such as Streptococcus pneumoniae contained numerous chains of gram-positive cocci. Brown and Benn stain, 1000× oil
and Streptococcus equi subsp. zooepidemicus. As is the case in magnification.

334
 CHAPTER 34  Streptococcal and Enterococcal Infections 335

streptococcal strains. Nevertheless, these traditional classifica- Streptococcus canis

tion methods are still used by laboratory staff, microbiologists,
and clinicians to describe streptococci.
Streptococci invade tissues opportunistically when there is a
breach in normal host barriers. This leads to a variety of clinical
disease manifestations, such as pyoderma, pneumonia, endo-
carditis, arthritis, osteomyelitis, meningoencephalitis, cellulitis,
and urinary tract infections (UTIs). Severe and life-threatening
manifestations of streptococcal infection include necrotizing
fasciitis (NF) and streptococcal toxic shock syndrome (STSS).
Predisposing conditions for streptococcal infections in dogs
and cats may include atopic dermatitis, wounds, foreign bodies
(such as migrating grass awns), immunosuppressive drug treat-
ment, and young age (especially neonates).3 The most common
clinical presentation among fetuses and neonates is streptococ-
cal bacteremia and sepsis, which can result in abortion and
death. Streptococcal pneumonia may occur in association with
infections with other respiratory pathogens, such as respiratory
viruses, or occur secondary to bronchitis, hematogenous spread
or aspiration. In one study, streptococci were isolated from sick
dogs more frequently in summer months.3
A B
FIGURE 34-2  Electron micrograph of Streptococcus pyogenes (A) and Streptococcus
canis (B). Proteinaceous cell surface fibrillae are present on S. canis that resemble the
M-protein surface fibrils of the human pathogen S. pyogenes. M-protein is a key virulence
factor of S. pyogenes. (From DeWinter LM, Low DE, Prescott JF. Virulence of Streptococcus
canis from canine streptococcal toxic shock syndrome and necrotizing fasciitis. Vet Micro-
biol 1999;70:95-110.)
S. canis is the most frequently isolated streptococcus from dogs
and cats.3 It is a β-hemolytic, group G (pyogenic) streptococ-
cus that colonizes the skin, genital, and gastrointestinal tracts
of healthy dogs and cats. It has also been isolated from other
animal species, such as rats, mice, rabbits, mink, and foxes.
Infection with S. canis may be associated with neonatal bacte-
remia, pharyngitis, cervical lymphadenitis, infective endocar-
ditis, UTIs, postoperative incision or wound infections, otitis
externa, keratitis, bronchopneumonia, pyometra or metritis,
meningoencephalitis, NF, STSS, rhinitis and necrotizing sinus-
itis, pyothorax, discospondylitis, arthritis, osteomyelitis, masti-
tis, cholangiohepatitis, and peritonitis. Neonatal infections can
occur when organisms are transmitted from the vaginal tract
during parturition. The organism can then gain access to the
systemic circulation via the umbilical vein. Streptococcal men-
ingitis results from direct extension from the sinuses or middle
ear, or from bacteremia. Other embolic complications may
accompany bacteremia with group G streptococci. Although
opportunistic infections with S. canis occur sporadically, out-
breaks of group G streptococcal infection have been reported
in group-housed animals, which suggested spread of a virulent
strain.4-6
Severe manifestations of S. canis infection, such as STSS and
NF, have been increasingly described in dogs and cats in recent
years, sometimes in the absence of obvious immunosuppressive
underlying conditions or wounds.6-9 Although toxic shock syn-
drome can also be caused by staphylococci, STSS is defined as
any streptococcal infection associated with the sudden onset of
shock and organ failure. Mechanisms of shock and organ fail-
ure identified in human group A streptococcal infections include
elaboration of pyrogenic exotoxins by streptococci, which act as
superantigens. Superantigens stimulate T cell responses through
their ability to bind to, and cross-link, the MHC class II com-
plex of antigen-presenting cells, and the T cell receptor, which
bypasses normal MHC-restricted antigen processing. This leads
to sudden and massive cascade of cytokine release, which in
turn causes signs of fever, vomiting, and hypotension, together
with tissue damage, disseminated intravascular coagulation

TABLE 34-1
Classification of Streptococci Isolated from Dogs and Cats
Lancefield Hemolytic
Species Antigen(s) Reactions Comments
S. canis G β Most common. Dogs and cats are natural hosts. Causes a variety
of clinical syndromes. Primary cause of streptococcal toxic shock
syndrome and necrotizing fasciitis in dogs and cats.
S. equi subsp. C β Causes pneumonia and sinusitis, primarily in group-housed animals.
zooepidemicus Has also been associated with meningitis.
S. dysgalactiae C β Uncommon. Has been isolated from dogs with septicemia, dermatitis,
or pneumonia.
S. bovis group D α or γ Uncommon. Has been isolated from dogs with urinary tract infections
(S. gallolyticus and and endocarditis.
S. infantarius)
S. agalactiae B β or γ Rare. Isolated from dogs and cats with endocarditis and neonatal
septicemia.
336 SECTION 2  Bacterial Diseases

(DIC), and multiple organ dysfunction.10 Other streptococcal
virulence factors also contribute to proinflammatory cytokine
release and the development of hypotension. Laboratory abnor-
malities include thrombocytopenia, azotemia, hypoalbumin-
emia, and metabolic acidosis. Death can occur within 48 hours
after the onset of illness. Specific criteria are used for diagno-
sis of STSS in human patients, and similar definitions could be
used for diagnosis of STSS in dogs and cats (Box 34-1). Because
only a few dogs and cats with STSS have been described, pre-
disposing factors have not been clearly identified. Predisposing
conditions in humans include diabetes mellitus, alcoholism, sur-
gical procedures, penetrating and nonpenetrating trauma, viral
infections such as varicella, and possibly the use of nonsteroidal
antiinflammatory drugs.
NF is a bacterial infection of the deep subcutaneous tissues
and fascia, characterized by extensive necrosis and gangrene
of the skin and underlying tissues. NF often begins as a minor
wound and progresses rapidly over 24 to 72 hours, and it may
be accompanied by STSS (Figure 34-3). The popular term
BOX 34-1
“flesh-eating bacteria” has been used to describe the organisms
involved. Streptococcal NF has been described in both dogs and
cats. Lesions usually involve a limb and are intensely painful,
with localized heat and swelling and accumulation of exudate
along fascial planes that requires drainage and debridement. In
some dogs, extensive sloughing of necrotic skin occurs.9 Nec-
rotizing myositis, bacteremia, and septic emboli may accom-
pany NF.8 Outbreaks of NF, arthritis, sinusitis, and meningitis
caused by S. canis have been reported in cats in shelters5 and
breeding colonies.11 Molecular typing (by pulsed-field gel elec-
trophoresis) of isolates from one outbreak revealed that iso-
lates were clonally related, which indicated spread of a virulent
strain.12
Despite the recognition of severe disease manifestations in
some dogs and cats, relatively little is known about virulence
factors of S. canis. A protein analogous to M protein, a major
virulence factor of S. pyogenes, has been identified in S. canis
and was shown to bind plasminogen and degrade thrombi.13
M protein has important antiphagocytic properties. Genes that
encode M protein and a streptococcal hemolysin, streptolysin
O, have been detected in S. canis isolates from dogs with NF
and STSS.14 Genes that encode other virulence factors identi-
fied in S. pyogenes, such as pyrogenic exotoxins (Spe genes),

Criteria Used for Diagnosis of STSS in Human Patients,

with Suggested Modifications for Diagnosis of STSS in Dogs
and Cats*

I  Isolation of Group G streptococci

(Streptococcus canis)
A. From a normally sterile site
B. From a nonsterile site

II  Clinical signs of severity

A. Hypotension: systolic blood pressure <90 mm Hg in
dogs and <80 mm Hg in cats
and
B. Two or more of the following signs:
1. Renal impairment: creatinine above the upper limit
of the reference range A
2. Coagulopathy: thrombocytopenia or DIC (prolonged
clotting times, low serum fibrinogen concentration,
and increased fibrin degradation products)
3. Liver involvement: serum AST, ALT, or total biliru-
bin levels at least twice the upper limit of the refer-
ence range
4. Acute respiratory distress syndrome defined as acute
onset of diffuse pulmonary infiltrates and hypox-
emia in the absence of cardiac failure, or evidence
of diffuse capillary leak manifested as acute onset of
pulmonary edema, or pleural or peritoneal effusions
with hypoalbuminemia
5. A generalized erythematous macular rash that may
desquamate
6. Soft tissue necrosis, including necrotizing fasciitis or
myositis, or gangrene B
FIGURE 34-3  A, Two-year-old female spayed domestic shorthair that was eutha-
*An illness that fulfills criteria IA and II is defined as a definite case. nized with necrotizing cellulitis, myositis and fasciitis, and peritonitis that developed after
An illness that fulfills criteria IB and II is defined as a probable case if an ovariohysterectomy site became infected with Streptococcus canis. B, Histopathology
no other cause for the illness is identified. showed severe necrotizing cellulitis, myositis, and cellulitis with intralesional cocci that
DIC, disseminated intravascular coagulation; STSS, streptococcal toxic sometimes formed chains. H&E stain, 1000× oil magnification. (Courtesy of the University
shock syndrome. of California, Davis, Veterinary Anatomic Pathology Service.)
 CHAPTER 34  Streptococcal and Enterococcal Infections
streptococcal superantigen (SSA), streptokinase (Ska), and C5a
peptidase (Scp, which cleaves the fifth component of comple-
ment) have not been detected.
Streptococcus equi subsp. zooepidemicus
S. equi subsp. zooepidemicus is a β-hemolytic, group C strepto-
coccus that is a commensal of the upper respiratory and lower
genital tracts of horses, and appears to be an emerging pathogen
of dogs and cats. Opportunistic infections such as wound infec-
tions, respiratory disease, endometritis, and abortion occur in
horses. Infections have also been reported in a variety of other
animal species, including cattle, sheep, pigs, monkeys, seals,
ruminants, dogs, cats, and humans. Different strains of S. equi
subsp. zooepidemicus exist that may vary in pathogenicity and
host tropism. Infected horses may have been a source of infec-
tion for dogs in some situations,15,16 but in other situations
where infected dogs and cats have been identified, a history of
horse contact was not present.
Several outbreaks of hemorrhagic, fibrinosuppurative, and
necrotizing pneumonia associated with S. equi subsp. zooepi-
demicus infection have been described in group-housed dogs,
such as those in kennels and shelter environments, from a vari-
ety of geographic locations worldwide (Figure 34-4).17 The
pneumonia progresses rapidly in some dogs can be accompa-
nied by signs suggestive of STSS, such as fever and systemic
hypovolemia. In some dogs, severe tachypnea and death occurs
within 48 hours of the first signs of respiratory disease, and dogs
may be found dead without any preceding clinical signs. Other
infected dogs show only mild signs of respiratory disease. S. equi
subsp. zooepidemicus bacteremia and sepsis has been reported
in racing greyhounds.18 Chronic lymphoplasmacytic rhinitis,
sometimes with turbinate lysis, has also been described in sev-
eral dogs in association with S. equi subsp. zooepidemicus infec-
tion, which resolved after specific antimicrobial treatment.15,16
Outbreaks of pneumonia, purulent rhinosinusitis, and menin-
goencephalitis have been described in cats.19,20 S. equi subsp.
FIGURE 34-4  Pneumonia in a shelter dog caused by Streptococcus equi subsp. zooepi-
demicus. The lungs are consolidated and dark red. (Courtesy of Dr. Patricia Pesavento, Uni-
versity of California, Davis.)
337
zooepidemicus meningoencephalitis occurred in a cat as a result
of extension of otitis media/interna.21
The pathogenesis of infection with S. equi subsp. zooepi-
demicus in dogs and cats is unclear. Co-infection with other
contagious respiratory pathogens can contribute to the sever-
ity of disease. Co-infections with canine distemper virus
(CDV), canine adenovirus-2 (CAV-2), canine herpesvirus-1
(CHV-1), canine influenza virus (CIV), Bordetella bronchi-
septica, or Mycoplasma cynos have been documented in
some, but not all, affected dogs with S. equi subsp. zooepi-
demicus pneumonia. Experimentally, co-infection of dogs
with both CIV and S. equi subsp. zooepidemicus is asso-
ciated with more severe clinical manifestations of disease
than when either organism is inoculated alone.22 Other
environmental factors such as overcrowding may also con-
tribute to stress and severe disease manifestations. Supe-
rantigen genes have been detected in some isolates of
S. equi subsp. zooepidemicus from dogs,23,24 but there has
been no correlation between the presence of these genes and
the severity of histopathologic lesions. However, expression
of proinflammatory cytokines such as TNF-α, Il-6, and Il-8 in
the lungs of affected dogs suggests that a potent inflammatory
response is involved in the pathogenesis of disease.23
Other Streptococcal Species
Like S. equi subsp. zooepidemicus, S. dysgalactiae is a β-hemolytic,
group C streptococcus. There are two subspecies, S. dysgalactiae
subsp. equisimilis and S. dysgalactiae subsp. dysgalactiae. After
S. canis and S. equi subsp. zooepidemicus, S. dysgalactiae subsp.
equisimilis was the third most common β- or γ-hemolytic strep-
tococcal species isolated from dogs at a veterinary teaching hos-
pital in the United States, and was detected in 13 (3.3%) of 393
dogs with streptococcal infections. Affected dogs had bacteremia
and sepsis, dermatitis, or pneumonia.3 S. dysgalactiae subsp. dys-
galactiae has been reported in association with systemic neonatal
infections and death in a litter of great Dane puppies.25
The porcine streptococcus, Streptococcus suis, has been
isolated from cats with meningoencephalitis, pyothorax, or
dermatitis.26,27 S. suis was also isolated from the urine of a sys-
temically unwell dog in Canada28 and can be isolated from the
tonsils, anus, and skin of healthy dogs and cats.29 Although con-
tact with pigs was not reported in the sick dogs and cats, the dog
was fed commercial pig ear treats. S. suis has been classified into
serotypes, which have epidemiologic importance. Human infec-
tions are usually serotypes 2 and 14. The cases reported in dogs
and cats have not belonged to these serotypes.
Other streptococcal species isolated from dogs and cats
include the human pathogen Streptococcus constellatus (a Strep-
tococcus anginosus group organism), which was isolated from a
dog with pyoderma;30 and group B streptococci (S. agalactiae),
which have been isolated from dogs and cats with streptococcal
disease such as endocarditis and neonatal sepsis. Group B strepto-
cocci are important causes of puerperal sepsis and neonatal sepsis
and meningitis in humans, as well as mastitis in cattle.
Streptococcus bovis Group (Streptococcus
gallolyticus and Streptococcus infantarius)
Streptococcus bovis group organisms are α- or nonhemolytic and
usually possess Lancefield group D antigens. Biotypes that belong
to this group, which differ in their biochemical characteristics,
have now been reclassified as distinct species, Streptococcus gallo-
lyticus and Streptococcus infantarius.31 S. bovis group organisms
338 SECTION 2  Bacterial Diseases
are commensals of the gastrointestinal tracts of humans and
other animals, especially ruminants. They are important causes
of bacteremia and endocarditis in humans, accounting for 11%
to 17% of all endocarditis cases. They frequently affect mul-
tiple valves and are associated with embolic complications. For
unknown reasons, fecal carriage of S. bovis group organisms by
humans has been strongly associated with colorectal cancer. This
especially seems to be true for S. gallolyticus subsp. gallolyticus.32
Rarely, S. bovis group organisms are isolated from dogs and cats
with UTIs. They have also been isolated from the blood of dogs
with mitral valve endocarditis, one of which also had sternebral
osteomyelitis.33,34
Enterococcus spp.
Enterococci are α- or nonhemolytic gram-positive bacteria that
usually possess Lancefield group D antigens, are commensals of
the gastrointestinal tracts of humans and other animals, and are
important nosocomial pathogens. They form pairs or short chains
of organisms and cannot morphologically be discriminated from
streptococci. Enterococci survive harsh environmental condi-
tions; they grow in medium that contains 6.5% NaCl, can grow
at temperatures that range from 10°C to 45°C (50°F to 113°F),
and hydrolyze esculin in the presence of 40% bile salts. The genus
Enterococcus was separated from Streptococcus in the mid-1980s
based on the results of DNA hybridization experiments.35
Enterococci are often resistant to a wide variety of different
antimicrobial drug classes, and multiple drug resistant (MDR)
enterococcal infections, especially those that are also resistant
to vancomycin (vancomycin-resistant enterococci or VRE),
are a significant problem in human medicine. Of great clinical
importance, resistance among enterococci occurs as a result of
both intrinsic and acquired resistance mechanisms. Intrinsic
resistance to low levels of most β-lactam antimicrobials occurs
because enterococci possess low-affinity penicillin binding pro-
teins (PBPs). Thus penicillins are generally bacteriostatic, not
bactericidal, against enterococci. Enterococci also have low-
level intrinsic resistance to aminoglycosides, which results from
decreased drug uptake. However, uptake of aminoglycosides
is enhanced when enterococci are exposed to β-lactams, which
explains the synergistic activity of this combination. Enterococci
are resistant to trimethoprim-sulfamethoxazole because they
utilize exogenously produced folate. Thus, even if the organ-
isms appear sensitive to trimethoprim-sulfamethoxazole in vitro
(when exogenous folate is lacking), the drug is not effective
in  vivo. Many laboratories therefore do not report minimum
inhibitory concentrations for this organism-drug combination.
Enterococcal resistance to other antimicrobials, such as mac-
rolides and vancomycin, can result from acquired resistance
mechanisms. Extensive multidrug resistance has been well doc-
umented in enterococci isolated from dogs and cats, but there
are only a few reports of enterococci with acquired vancomycin
resistance from dogs in the literature. These have been from the
United Kingdom, New Zealand, and the United States.36-38 All
other isolates examined have been vancomycin susceptible. In
contrast, VRE account for about 30% of enterococcal infec-
tions in humans from Europe and North America.39
Clinical microbiology laboratories often do not differentiate
between Enterococcus species, because definitive identification
can require genetic typing. Some laboratories use biochemical
methods to identify Enterococcus faecalis and Enterococcus
faecium, which are the most prevalent species in humans and
which differ in their epidemiology, antimicrobial susceptibility,
and virulence properties. E. faecium is more likely to show high-
level resistance to penicillins and carbapenems than E. faecalis,
and as a result, in humans, the prognosis for serious E. faecium
infections may be worse than that for E. faecalis infections.40
However, E. faecalis is more likely to produce biofilms than
E. faecium.41 Although most infections in humans have been
E. faecalis, a worrisome increase in the prevalence of MDR E.
faecium infections has occurred. In particular, ampicillin- and
fluoroquinolone-resistant E. faecium strains that belong to hos-
pital adapted strains known as clonal complex 17 (CC17) have
emerged. CC17 isolates have been found in dogs, but their viru-
lence gene profile and antimicrobial susceptibility profiles have
differed from those of human isolates.42
In healthy dogs and cats, enterococci can be found on the
skin and within the oral cavity, nasal cavity, and gastrointesti-
nal tract. In the United States, the most prevalent species in dogs
appears to be E. faecalis, which comprised 68% of 155 iso-
lates.43 In the cat, it was Enterococcus hirae, which comprised
52% of 121 isolates.43 Preparations that contain E. faecium
strain SF68 have been investigated as probiotics in dogs and
cats, with mixed results.44-46 Some studies suggest that supple-
mentation of food with this probiotic can stimulate local and
systemic immune function in dogs and cats.47,48
Although less pathogenic than many streptococci, impor-
tant virulence factors have been identified in enterococci that
enable them to invade tissues and cause disease. These include
a cytolysin, gelatinase, and a serine protease, as well as several
surface proteins (e.g., the Ace protein, which allows the organ-
ism to adhere to connective tissue components, such as colla-
gen). Enterococci also possess pili that play an important role
in adherence. The ability of many enterococci to form biofilms
means they can be very difficult to eradicate, because they resist
phagocytosis and antimicrobial drugs.41 As in humans, the two
most common species of Enterococcus associated with disease
in dogs and cats are E. faecalis and E. faecium.36,49 Others are
shown in Box 34-2.
BOX 34-2
Enterococcus Species Isolated from Healthy and Sick
Dogs and Cats36,43,50,51
Healthy Dogs and Cats
E. faecalis
E. faecium
E. hirae
E. casseliflavus
E. durans
E. canintestini
E. avium
Sick Dogs and Cats
E. faecalis
E. faecium
E. hirae
E. casseliflavus
E. durans
E. gallinarum
 CHAPTER 34  Streptococcal and Enterococcal Infections
Because enterococci are naturally resistant to a number
of antimicrobials, treatment with broad-spectrum antibiotics
(which destroy competing bacteria within the gastrointestinal
tract) selects for colonization by enterococci. The development
of enterococcal UTIs may be promoted by repeated use of anti-
microbial drugs to treat recurrent UTIs, when the underlying
cause of infection is not or cannot be resolved. Enterococci can
contaminate the hospital environment and survive for long peri-
ods on fomites such as doorknobs, stethoscopes, and monitoring
devices, as well as the hands, gloves, and clothing of hospital per-
sonnel. Inoculation may occur via urinary, intravenous, or other
invasive devices, or organisms may enter the body from a dam-
aged gastrointestinal tract. Severe concurrent illness and immuno-
suppression may predispose to infection. In addition to skin and
UTIs, enterococci may be isolated from dogs and cats with UTIs,
pyoderma and otitis externa, cholangiohepatitis, pancreatitis,
hepatic abscesses, peritonitis, mastitis, bacteremia and endocardi-
tis, wound infections, and discospondylitis. There have been rare
reports of gastrointestinal illness in association with enterococcal
infection of the gastrointestinal tract in dogs and cats. In affected
cats, an association with diarrhea was supported by the presence
of large mats of enterococci in association with the gastrointesti-
nal mucosa.52,53 Frequently, enterococci are isolated from lesions
in mixed infections with other gastrointestinal microorganisms.
Physical Examination Findings
Physical examination findings in dogs and cats with Streptococ-
cus or Enterococcus infections vary with the anatomic site(s) of
infection. Streptococcal infections are an important differential
diagnosis in sick or fading neonates; dogs that have physical
examination findings suggestive of endocarditis; dogs and cats
with severe pneumonia and pyothorax; NF; rapid onset of sys-
temic illness in association with a wound; or outbreaks of severe
purulent upper respiratory disease, hemorrhagic pneumonia,
and systemic illness in group-housed animals.
Findings suggestive of streptococcal or enterococcal endocar-
ditis include fever, lethargy, tachycardia, joint pain and swelling,
systolic heart murmurs, and signs of embolic complications such as
lameness, limb edema, weak or absent pulses, or neurologic signs.
Animals with streptococcal pneumonia may have a fever (up to
106.3°F or 41.3°C), cough, tonsillar enlargement and hyperemia,
mucopurulent nasal discharge, and/or tachypnea with increased
lung sounds. Lung sounds may be decreased if pleural effusion is
present. Animals with NF have variable extents of regional cutane-
ous erythema, sometimes in association with a wound; cutaneous
ulceration, swelling, edema, intense pain, and warmth on palpa-
tion of the affected area. In some animals, extensive skin sloughing
occurs. Animals with STSS may be obtunded, with weak pulses,
tachycardia, tachypnea, and prolonged capillary refill time. Rectal
temperatures may exceed 106°F (41.1°C), although the presence
of fever is variable. Icterus may be present in animals that develop
multiple organ dysfunction syndrome in association with STSS.
Diagnosis
Laboratory Abnormalities
Complete Blood Count, Biochemistry, and Urinalysis Findings
Dogs and cats with severe manifestations of streptococcal infec-
tions, such as pneumonia, NF, endocarditis, and STSS, usually
have a neutrophilic leukocytosis with a left shift, toxic neutro-
phils, lymphopenia, and monocytosis. A degenerative left shift
or circulating metamyelocytes may be present. STSS may be
339
accompanied by thrombocytopenia as a result of DIC, azote-
mia and/or increased liver enzyme activities, hypoalbuminemia,
and hypoglycemia. The urinalysis of dogs and cats with strep-
tococcal or enterococcal UTIs may reveal pyuria, proteinuria,
or hematuria, and cocci in pairs or chains may sometimes be
evident on sediment examination.
Clotting Function
Dogs and cats with STSS that develop DIC may have clotting
function abnormalities including prolonged coagulation times,
increased fibrin degradation product and D-dimer concentra-
tions, and decreased antithrombin concentrations.
Cytologic Abnormalities
Cytologic examination of specimens (respiratory lavage fluid,
aspirates of affected tissues, or pus) from dogs and cats with
streptococcal or enterococcal infections typically reveals large
numbers of degenerate and nondegenerate neutrophils. Coccoid
bacteria in pairs or chains may also be seen.
Microbiologic Tests
Isolation and Identification
Streptococci and enterococci that infect dogs and cats are read-
ily isolated on routine bacteriologic media from aspirates, tis-
sue biopsies, body fluids, and lavage specimens. Inoculation of
complex media that contain blood is recommended. Streptococci
grow well in anaerobic incubation conditions, but these are not
required because of their facultative nature. The laboratory may
use selective media for specimens that are heavily contaminated
with other bacteria. Species are identified based on their pheno-
typic characteristics (such as colony morphology and hemolytic
properties), biochemical reactions, and serotyping with Lance-
field antisera; the last is available on a commercial basis. As
noted previously, genetic typing may be required to identify some
enterococci to the species level. Molecular typing methods such
as pulsed-field gel electrophoresis may be required to investigate
outbreaks in group-housed animals or hospital environments.
Antimicrobial susceptibility testing for streptococci is often
not performed by veterinary diagnostic laboratories, because vir-
tually all strains are penicillin susceptible. Group A streptococci
(GAS) that infect humans remain universally susceptible to peni-
cillin, but an increased prevalence of penicillin resistance, which
occurs as a result of altered PBPs, has been detected in S. pneu-
moniae, which is primarily a human pathogen.54 S. pneumoniae
may also be resistant to other β-lactam drugs and macrolides.
Evaluation of antimicrobial susceptibility is of critical importance
for enterococci, because of the high prevalence of resistance in
this group, and especially among E. faecium isolates. Susceptibil-
ity testing for aminoglycosides requires high-concentration disk
diffusion or broth microdilution methods, which may not rou-
tinely be performed by some veterinary microbiology laboratories
(see Treatment).
Pathologic Findings
Pathologic findings in Streptococcus infections vary depending
on the organ system affected. Gross findings in S. canis sinusitis
include accumulation of purulent material within the frontal
sinus with osteolysis, and distortion of the nasal bridge in cats.5
When meningitis is present, purulent material may be found
in association with the meninges (Figure 34-5). Lesions of NF
in dogs and cats consist of edema, ecchymoses, and necrosis
that extend within the fascia and subcutis in association with
340 SECTION 2  Bacterial Diseases
FIGURE 34-5  Accumulation of purulent exudate over the meninges in a shelter cat
that died of Streptococcus canis meningitis. (Courtesy of Dr. Patricia Pesavento, University
of California, Davis.)
ulcerative skin lesions (see Figure 34-3). An accumulation of
pus may be found between fascial planes. Congestion of a
variety of organs may be present in animals that have accom-
panying STSS. When S. canis or Enterococcus endocarditis is
present, adherent and friable mass lesions may be seen in asso-
ciation with the mitral and/or, less commonly, the aortic valves.
Dogs with streptococcal endocarditis are more likely to have
concurrent polyarthritis than dogs with other bacterial causes
of endocarditis.33 On histopathology, valve lesions generally
contain large numbers of degenerate and nondegenerate neu-
trophils, hemorrhage, necrosis, fibrin thrombi, and abundant
gram-positive cocci (Figure 34-6). Septic thromboemboli may
be present in a variety of organs. Histopathologic lesions in
neonates that die of neonatal sepsis include severe inflamma-
tion and necrosis, as well as septic emboli in a variety of tissues
(see Figure 34-1).
Grossly, the lungs of dogs with S. equi subsp. zooepidemicus
pneumonia may be red, consolidated, and fail to collapse (see
Figure 34-4). There may be variable quantities of hemorrhagic
pleural effusion, petechial and ecchymotic hemorrhages on pleu-
ral surfaces, and bloody exudate within the airways and nasal
cavity.17 Typical histopathologic changes include fibrinous, nec-
rotizing, and/or suppurative pneumonia or bronchopneumonia;
fibrin thrombi; and intralesional clusters or chains of coccoid
bacteria. Within lymph nodes, marked lymphoid depletion may
be present, but lymphoid hyperplasia has also been reported.
Occasionally, septic thromboemboli may be present in organs
such as the lymph nodes, renal glomeruli, adrenal glands, brain,
and splenic sinusoids.55,56
Treatment and Prognosis
In general, streptococcal infections should be treated with a
β-lactam drug, preferably a penicillin or a cephalosporin (Table
34-2). Early diagnosis and prompt treatment with appropriate
antimicrobial drugs is essential to prevent more severe compli-
cations such as NF or STSS. In animals that present with severe
signs such as septic shock or life-threatening pneumonia, initial
treatment should be with a broad-spectrum antimicrobial drug
combination, such as a β-lactam and an aminoglycoside, which
FIGURE 34-6  Histopathology of the mitral valve of an 8-year-old male neutered
springer spaniel that was seen for a 1-month history of right thoracic limb lameness,
anorexia, and lethargy. There is severe, subacute, fibrinonecrotic endocarditis with intra-
lesional cocci. When the dog was first evaluated, the platelet count was 9000 platelets/
µL and the dog was treated with vincristine for immune-mediated thrombocytopenia.
Subsequently the dog developed vomiting and diarrhea and became obtunded. Blood
cultures grew Streptococcus canis. At necropsy, septic emboli were present in the kidneys,
spleen, and lymph nodes. Necrotizing fasciitis was also present in multiple limbs, which
was thought to have developed as a result of septic embolization.
may also have synergistic activity against some streptococci,
such as group C streptococci (S. equi subsp. zooepidemicus).
Necrotizing Fasciitis, Myositis,
and Toxic Shock Syndrome
Prompt surgical exploration and debridement is of critical
importance in NF, because shock and organ failure will con-
tinue to progress in the face of antimicrobial treatment when
necrotic tissue persists. Imaging techniques such as ultrasound
may be useful to help characterize the extent of lesions before
surgery. Extremely aggressive fluid resuscitation with crystal-
loids and blood products may also be required, while blood
pressure is monitored. In human patients, deep-seated strep-
tococcal infections (NF and STSS) are treated with both high-
dose penicillin and clindamycin. This is because clindamycin
suppresses exotoxin production by GAS, is more active than
penicillin in experimental NF, and has a longer half-life than
penicillin. However, clindamycin resistance exists in some GAS,
whereas all GAS are susceptible to penicillin.57 Also, penicil-
lin may not be effective in deep-seated streptococcal infections,
because stationary-phase organisms do not express PBPs. This
tolerance to penicillin has also been observed in group G strep-
tococcal infections.58 Approximately 10% to 15% of contem-
porary S. canis isolates from dogs in Europe have demonstrated
clindamycin resistance.59,60 Given this finding, the same combi-
nation could be used to treat NF in dogs and cats.
Meningitis/Meningoencephalitis
Although high-dose penicillin has been used to treat strepto-
coccal meningitis in human patients, penicillins typically have
limited penetration of the CSF. Trimethoprim-sulfamethoxazole
was used with apparent success to treat some dogs and cats with
streptococcal meningitis.21,61 See Chapter 90 for additional
 CHAPTER 34  Streptococcal and Enterococcal Infections 341

TABLE 34-2
Suggested Antimicrobial Drugs for Treatment of Streptococcal Infections in Dogs and Cats
Drug Dose (mg/kg) Interval (hours) Route
Penicillin G potassium or sodium* 20,000-40,000 U/kg 6-8 IV, IM
Ampicillin sodium* 10-20 6-8 IV, IM, SC
Amoxicillin 6.6-20 8-12 PO
Cefazolin sodium 20-35 8 IV, IM
Cephalexin 10-30 (dogs), 15-20 (cats) 12 PO
Trimethoprim-sulfamethoxazole† 30 12 PO, IV

*Consider use with clindamycin, 10 mg/kg q12h IV or IM for necrotizing fasciitis or myositis; consider addition of gentamicin sulfate, 9-14 mg/kg
q24h IV, IM, or SC for dogs with S. equi subsp. zooepidemicus pneumonia because of synergy of this combination for group C streptococci.
†Consider use for treatment of streptococcal meningitis.

TABLE 34-3
Suggested Medications for Treatment of Enterococcal Infections in Dogs and Cats
Drug Dose (mg/kg) Interval (hours) Route
Ampicillin sodium* 10-20 6-8 IV, IM, SC
Amoxicillin* 6.6-20 8-12 PO
Vancomycin† 15 8 IV
Linezolid† 10 (dogs) 8 PO
Chloramphenicol 40-50 (dogs), 12.5-20 (cats) 6-8 (dogs), 12 (cats) PO, IV, IM
Nitrofurantoin‡ 2-3 8 PO

*In human patients, used with gentamicin sulfate, 1 mg/kg q12h IV for systemic enterococcal infections caused by enterococci susceptible to high-
level aminoglycosides, because of the synergy of this combination
†For enterococci that show high-level aminoglycoside resistance or when aminoglycosides cannot be used because of toxicity (animals with renal

failure). Use in veterinary patients is controversial. Refer to Chapter 8 for additional information on the use of vancomycin.
‡For resistant UTIs

information on treatment of bacterial meningitis. Ceftriaxone
has been used as an alternative to benzylpenicillin for treatment
of human streptococcal meningitis and attains higher concen-
trations in the CSF.
Bacteremia and Endocarditis
The reader is referred to Chapter 86 for information on treat-
ment of bacteremia and endocarditis.
Enterococcal Infections
Suggested medications for treatment of enterococcal infections in
dogs and cats are shown in Table 34-3. Successful treatment of
enterococcal infections requires knowledge of the intrinsic resis-
tance of enterococci to low levels of β-lactams and aminoglyco-
sides, the synergistic effect of high doses of these drugs, the lack
of efficacy of trimethoprim-sulfonamide combinations in  vivo,
and the ability of enterococci to acquire resistance genes to a
large number of other antimicrobial compounds. Aminopenicil-
lins such as ampicillin generally have more potent activity against
enterococci than does penicillin or the carbapenems. Bactericidal
regimens, which consist of a β-lactam and gentamicin (or strep-
tomycin), are used in human patients to treat systemic infections
such as endocarditis or bacteremia. Enterococci are usually
resistant to aminoglycosides at dilutions used in routine broth
microdilution methods (low-level resistance). When the synergis-
tic combination is indicated, but low-level resistance is present,
isolates should be tested for high-level resistance (HLR) to ami-
noglycosides. Aminoglycoside HLR results from production of
an aminoglycoside-modifying enzyme by enterococci. It emerged
in the 1980s and has been a growing problem in both E. faecium
and E. faecalis isolates from human patients. Aminoglycoside
HLR has also been reported in E. faecium and E. faecalis iso-
lated from dogs and cats,36,38,62 but most isolates are susceptible
to high concentrations of gentamicin. In human patients, a com-
bination of ceftriaxone and ampicillin has been recommended to
treat endocarditis caused by aminoglycoside HLR enterococci.39
When enterococci show penicillin resistance, vancomycin can be
substituted for penicillin, but this requires prolonged hospitaliza-
tion for intravenous administration. Single-agent treatment with
linezolid (see Chapter 8) is an oral alternative for treatment of
serious penicillin-resistant enterococcal infections, but in human
patients it is recommended that this only be used when combi-
nations of β-lactams and gentamicin cannot be used because of
resistance, toxicity, or treatment failure.39 Although most strains
342 SECTION 2  Bacterial Diseases
of enterococci isolated from humans are susceptible to line-
zolid, resistant strains have appeared.63 Chloramphenicol may
be successful for treatment of resistant enterococcal bloodstream
infections when administered orally, but it is bacteriostatic, and
concerns regarding adverse effects exist (see Chapter 8).
Treatment of enterococcal UTIs with β-lactams such as
ampicillin or amoxicillin may be successful even when resis-
tance is documented in the susceptibility panel, because of the
high concentrations that these drugs achieve in the urine.39
Nitrofurantoin has also been useful for treatment of resistant
enterococcal UTIs in humans, and routine treatment of subclini-
cal bacteriuria caused by MDR enterococci is not recommended
(see Chapter 89).64 When enterococci are present with other
bacteria, especially in the urinary tract, treatment of the nonen-
terococcal pathogen may be sufficient to resolve the infection.
Prevention
Because outbreaks of streptococcal infections in dogs and cats
have been associated with overcrowding, co-infections, and
other stressors, appropriate shelter and kennel management and
hygiene, as well as vaccination against other respiratory patho-
gens, may help to prevent streptococcal infections. Proper wound
care may prevent serious streptococcal infections such as bactere-
mia, endocarditis, NF, and STSS. Methods to reduce nosocomial
infections by enterococci are outlined in Chapter 11. Resistant
enterococcal infections may be prevented through restricted and
appropriate use of antimicrobial drugs and proper management
of underlying disorders that predispose to bacterial infections.
Public Health Aspects
Streptococci include some of the most important bacterial
pathogens of humans. The β-hemolytic group A streptococ-
cus (GAS) S. pyogenes causes streptococcal pharyngitis (“strep
throat”) and scarlet fever, as well as poststreptococcal rheu-
matic fever, rheumatic heart disease, and glomerulonephritis.
It has been estimated that globally, GAS cause 616 million
new cases of pharyngitis each year.65 GAS has also been
associated with other disease manifestations, which include
NF, STSS, myositis, vaginitis, and bacteremia. As a result,
S. pyogenes is one of the most extensively studied of all strep-
tococci. There are more than 100 different serotypes, which
are classified based on the sequence of the M protein gene
(known as emm typing). M protein is a filamentous macromol-
ecule that traverses the cell wall and is a key virulence factor of
GAS. Transmission between humans primarily occurs through
CASE EXAMPLE
Signalment: “Jazz” a 6-year-old female spayed miniature
Pinscher from Fairfield in northern California
History: Jazz was brought to the University of California,
Davis, Veterinary Medical Teaching Hospital for evaluation
of anorexia, lethargy, and diarrhea. Two days previously,
the dog had been seen by the dermatology service for a
focal area of alopecia and scaling on the dorsal midline in
close contact, and chronic subclinical carriage of infection in
the oropharynx occurs in some individuals. Although family
pets have been implicated as a reservoir in households with
recurrent streptococcal pharyngitis,66,67 colonization or infec-
tion of dogs and cats with GAS appears to be rare or nonexis-
tent when accurate streptococcal identification methods have
been performed. One study found no evidence of oropharyn-
geal colonization by GAS in pets that were in contact with
children with acute pharyngitis.68 Even among human family
members who are in contact with children with streptococcal
pharyngitis, routine culture of asymptomatic individuals is not
recommended, because penicillin fails to eradicate streptococ-
cal pharyngeal carriage.57
The α-hemolytic streptococcus S. pneumoniae is a commensal
of the human respiratory tract and can invade opportunistically
to cause a variety of clinical manifestations that include otitis
media, sinusitis, meningitis, bacteremia, and pneumonia. It pos-
sesses no Lancefield group antigens. Although extremely rare in
dogs and cats, infections with S. pneumoniae have been reported
in a dog with meningoencephalitis,61 a cat with arthritis,69 and a
cat with cellulitis.70 It is likely that in these reports, the dog and
cats acquired the infection from humans, because humans are the
natural hosts for S. pneumoniae.
Reports of human infection with S. canis and S. equi subsp.
zooepidemicus exist. S. canis has increasingly been recognized as a
cause of soft tissue infections in dog owners and has been isolated
from humans with bacteremia, UTIs, osteomyelitis, and pneumo-
nia.71,72 Rarely, S. equi subsp. zooepidemicus has been isolated
from humans with meningitis, bacteremia, endocarditis, pneu-
monia, osteomyelitis, septic arthritis, acute nephritis, or STSS.
Most human infections with S. equi subsp. zooepidemicus have
been traced to an animal source, such as contact with horses, or
consumption of inadequately pasteurized milk products or pork.
However, apparent transmission of S. equi subsp. zooepidemicus
from a dog with S. equi subsp. zooepidemicus pneumonia to a han-
dler, who developed severe systemic illness, has been described.73
Dogs and cats are also a potential source for human infection
by MDR enterococci. In one study, dogs that left a veterinary
intensive care unit were colonized with MDR enterococci with
the capacity for biofilm formation and horizontal transfer of
antimicrobial resistance genes.74
Because of the potential for transmission of streptococci and
enterococci between dogs, cats, and humans, preventative mea-
sures such as hand washing after pets are handled, household
disinfection, and avoidance of close contact (such as allowing
pets to lick wounds or mucous membranes) should be consid-
ered, especially for immunocompromised pet owners.
the thoracolumbar region that first appeared 7 months
previously, enlarged to 6 × 4 cm in diameter over 2 months,
and then remained static in size for the subsequent 5 months.
Skin scrapings and fungal culture were performed, and four
skin biopsies were obtained. An underlying vasculitis was
suspected, and treatment with vitamin E (200 U PO q12h)
and an oral fatty acid supplement was recommended
pending the results of the diagnostic tests. The following
day the owner noted edema of the right pelvic limb and
discontinued the medications. Over the next 24 hours, the
 CHAPTER 34  Streptococcal and Enterococcal Infections
dog became lethargic, inappetent, and developed liquid
diarrhea and polydipsia. Jazz had been vaccinated regularly
for canine distemper, adenovirus and parvovirus infections
and rabies and normally ate a commercial dry dog food. She
was last vaccinated just after the skin lesion first appeared.
The dog was mostly indoors, going outside on leash walks
only, and there were no other pets in the household.
Other Medical History: Jazz had been spayed 5 weeks
previously, at which time pyometra was an incidental
finding and was treated with a 7-day course of amoxicillin.
Physical Examination:
Body Weight: 3.3 kg
General: Lethargic, alert and ambulatory, but weak. Vocalized
on palpation of the thorax and abdomen. T = 102.2°F
(39.0°C), HR = 120 beats/min, RR = 60 breaths/min, mucous
membranes pink, CRT = 1 s.
Eyes, Ears, Nose, and Throat: Mild conjunctival hyperemia
and bilateral serous ocular discharge were present.
Integument: There was mild subcutaneous edema and a small
amount of serosanguineous fluid in the region of the skin
biopsies, but sutures remained intact. There was a single,
raised erythematous lesion adjacent to the skin biopsies
that measured 0.5 × 0.5 cm in diameter. Severe edema was
noted in the cranioventral thoracic region, which extended
over an area that was 9 × 5 cm in diameter.
Other Systems, Including Peripheral Lymph Nodes: No
clinically significant findings.
Laboratory Findings:
CBC:
HCT 53.1% (40%-55%)
MCV 71.2 fL (65-75 fL)
MCHC 36.7 g/dL (33-36 g/dL)
Nucleated RBC 24/100 WBC
WBC 2200 cells/µL (6000-13,000 cells/µL)
Neutrophils 792 cells/µL (3000-10,500 cells/µL)
Band neutrophils 748 cells/µL
Metamyelocytes 88 cells/µL
Myelocytes 44 cells/µL
Lymphocytes 308 cells/µL (1000-4000 cells/µL)
Monocytes 220 cells/µL (150-1200 cells/µL)
Platelets 97,000/µL (150,000-400,000 platelets/µL).
Markedly toxic band neutrophils, metamyelocytes, and
myelocytes; moderately toxic neutrophils; and rare reac-
tive lymphocytes were present.
Serum Chemistry Profile:
Sodium 128 mmol/L (145-154 mmol/L)
Potassium 4.4 mmol/L (3.6-5.3 mmol/L)
Chloride 90 mmol/L (108-118 mmol/L)
Bicarbonate 21 mmol/L (16-26 mmol/L)
Phosphorus 8.7 mg/dL (3.0-6.2 mg/dL)
Calcium 10.0 mg/dL (9.7-11.5 mg/dl)
BUN 63 mg/dL ( 5-21 mg/dL)
Creatinine 0.9 mg/dL (0.3-1.2 mg/dL)
Glucose 75 mg/dL (64-123 mg/dL)
Total protein 5.6 g/dL (5.4-7.6 g/dL)
Albumin 2.2 g/dL (3.0-4.4 g/dL)
Globulin 3.4 g/dL (1.8-3.9 g/dL)
ALT 185 U/L (19-67 U/L)
AST 128 U/L (19-42 U/L)
ALP 539 U/L (21-170 U/L)
343
Cholesterol 526 mg/dL (135-361 mg/dL)
Total bilirubin 0.5 mg/dL (0-0.2 mg/dL)
Urinalysis: SGr 1.021; pH 5.0, 1+ protein (SSA), 1+ bilirubin, 4+
hemoprotein, no glucose, no ketones, rare WBC/HPF, 0-2
RBC/HPF, few amorphous crystals, no bacteria seen
Imaging Findings: Abdominal ultrasound: Enlargement of
the uterine stump was present dorsal to the bladder. There
were no cystic structures within the stump and no evidence
of fluid in this region or lymphadenopathy.
Cytologic Findings: Needle aspiration of edema fluid from a
region adjacent to the right axilla revealed large numbers of
cocci, some in pairs, in the absence of significant numbers
of leukocytes.
Microbiologic Testing: Aerobic and anaerobic blood
culture (three isolator specimens): One colony of coagulase-
negative staphylococci was grown on one plate. The other
two plates were negative. No anaerobes were cultured.
The isolate was resistant to penicillin G, erythromycin, and
trimethoprim-sulfamethoxazole, but susceptible to all other
antimicrobials tested.
Aerobic bacterial culture (aspirate of edema fluid): Large num-
bers of Streptococcus canis colonies.
Treatment and Outcome: Treatment with IV fluids (0.9%
NaCl with 20 mEq KCl/L) was initiated. Ampicillin (20 mg/kg IV
q6h), enrofloxacin (5 mg/kg IV q24h), and butorphanol were
administered. The dog’s body temperature subsequently
dropped to 98.0°F (36.7°C) and blood glucose concentration
dropped to 30 mg/dL. Fluids were supplemented with
dextrose and a warming pad was used, and the dog’s
temperature returned to normal limits. Within hours the
edema had spread to the abdomen, and evidence of
bruising appeared on the right thorax and ventrum. The
raised erythematous lesion on the dorsum ulcerated, and
dehiscence of the biopsy site adjacent to this region became
apparent. Blood work showed a hematocrit of 39%, 2700
white cells/µL, 1323 band neutrophils/µL, 702 neutrophils/µL,
162 lymphocytes/µL, 513 monocytes/µL, and 37,000 platelets/
µL; serum sodium concentration was 142 mmol/L, potassium
3.7 mmol/L, creatinine 0.4 mg/dL, BUN 23 mg/dL, albumin
1.2 g/dL, globulin 3.1 g/dL, cholesterol 221 mg/dL, and total
bilirubin 2.3 mg/dL. The activities of ALT, AST, and ALP were 79
U/L, 136 U/L, and 292 U/L, respectively. Treatment with dextran
and plasma was initiated, but systolic blood pressure dropped
to 80 mm Hg, and the region adjacent to the biopsy sites
became firm and painful and tore when palpated. Ampicillin
was substituted with penicillin G (40,000 U/kg IV q6h). Forty-
eight hours after admission, the dog was anesthetized and a
large portion of necrotic skin was surgically debrided from the
dog’s dorsum. The debrided region was covered with a stent.
During recovery the dog was tachypneic, had increased lung
sounds, and systolic blood pressure dropped to 50 mm Hg.
The dog developed respiratory arrest, and an endotracheal
tube was placed, from which bloody fluid emanated. Cardiac
arrest then ensued, and there was no response to closed
cardiopulmonary resuscitation efforts.
Necropsy Findings: At necropsy, necrosis extended
through the intercostal musculature of the right thoracic
wall. Histopathologic lesions included severe, focally
extensive, hemorrhagic and necrotizing panniculitis and
fasciitis with intralesional bacteria and multifocal thrombi;
Continued
344 SECTION 2  Bacterial Diseases
diffuse necrotizing myositis of the right thoracic wall with
intralesional bacteria; severe congestion and mild, multifocal
hemorrhage and thrombosis within the kidneys; centrilobular
congestion and severe bile stasis within the liver; congestion
and hemorrhage within the lung; and generalized lymphoid
depletion and sinusoidal histiocytosis within lymph nodes.
Around the uterine stump, there was focal fibrosis and mild
granulomatous serositis. Very small numbers of S. canis were
cultured from the uterine stump. Histopathology of the biopsy
specimens that had been collected before the onset of systemic
illness showed severe, diffuse adnexal atrophy; mild, chronic,
lymphoplasmacytic mural folliculitis; focal granulomatous
furunculosis with hyperkeratosis; and mild, multifocal,
SUGGESTED READINGS
Ghosh A, Dowd SE, Zurek L. Dogs leaving the ICU carry a very
large multi-drug resistant enterococcal population with capac-
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2011;6:e22451.
Lappin E, Ferguson AJ. Gram-positive toxic shock syndromes. Lancet
Infect Dis. 2009;9:281-290.
Prescott JF, DeWinter L. Canine streptococcal toxic shock syndrome
and necrotising fasciitis. Vet Rec. 1997;140:263.
Priestnall S, Erles K. Streptococcus zooepidemicus: an emerging canine
pathogen. Vet J. 2011;188:142-148.
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346 SECTION 2  Bacterial Diseases
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for biofilm formation and horizontal gene transfer. PLoS One.
2011;6:e22451.
CHAPTER 35
Staphylococcus Infections
Jane E. Sykes
Overview of Staphylococcal Infections
in Dogs and Cats
First Described: 1882 in Scotland (Alexander Ogsten)1
Causes: In dogs and cats, the most common causes are Staph-
ylococcus pseudintermedius, Staphylococcus schleiferi,
Staphylococcus aureus, and various coagulase-negative
staphylococci, which are gram-positive coccoid bacteria.
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact (or through fomi-
tes). Organisms are often commensals that invade
opportunistically.
Major Clinical Signs: Pyoderma, otitis externa, surgical site
infections, osteomyelitis, bacteremia and endocarditis,
bronchopneumonia, urinary tract infections, ocular surface
infections, rarely necrotizing fasciitis and toxic shock syn-
drome, pyothorax and peritonitis, discospondylitis, arthritis
Differential Diagnoses: Primarily other gram-positive and gram-­
negative bacterial infections
Human Health Significance: Humans can become colonized
with staphylococcal isolates that colonize or infect dogs
and cats. Methicillin-resistant S. aureus, which can persis-
tently colonize humans, is of greatest concern, although
colonization of dogs with MRSA appears to be transient.
S. pseudintermedius, which persistently colonizes dogs, has
occasionally been isolated from sick and healthy people
who are in contact with dogs, and is of concern because of
its tendency to show a high degree of multidrug resistance.
Etiology and Epidemiology
Staphylococcus spp. are gram positive, nonmotile, non–spore
forming, and usually catalase-positive cocci, which occur sin-
gly and in pairs or grapelike clusters (Figure 35-1). Most spe-
cies are facultative anaerobes. Staphylococci colonize the skin
and mucous membranes of an enormous variety of animal spe-
cies, and disease generally follows a breakdown in normal host
defenses. Staphylococci can also contaminate surfaces in the
environment, where they may survive for several months.
Staphylococci are classified as coagulase negative or coag-
ulase positive based on their ability to produce coagulase, an
enzyme that cleaves fibrinogen into fibrin and results in coagu-
lation of plasma. There are more than 40 species in the genus
Staphylococcus,2 many of which are adapted to live on certain
host species. The major coagulase-positive staphylococci that
infect dogs and cats are Staphylococcus pseudintermedius (pre-
viously identified as Staphylococcus intermedius) and Staphylo-
coccus aureus. Staphylococcus schleiferi has also been isolated
from dogs (and rarely cats) with pyoderma and otitis externa
and has been divided into S. schleiferi subsp. schleiferi, which
is coagulase-negative, and S. schleiferi subsp. coagulans, which
is coagulase-positive. However, these may instead represent a
single species, S. schleiferi, with variable coagulase production.3
Coagulase-negative staphylococci (CoNS) tend to be of lower
virulence than coagulase-positive staphylococci but occasion-
ally cause disease in immunocompromised hosts. Examples of
coagulase-negative staphylococci are Staphylococcus felis and
Staphylococcus epidermidis. S. felis has been recognized as a
urinary tract pathogen in cats.4 CoNS are often not identified to
the species level by microbiology laboratories.
In recent years, the prevalence of antimicrobial drug resistance
has increased in Staphylococcus isolates obtained from canine and
feline infections. A significant proportion of resistant isolates pos-
sess the mecA gene, which encodes an altered penicillin binding
protein (PBP), known as PBP2a. The mecA gene is located on a
large genetic element, the staphylococcal cassette chromosome.
This PBP has low affinity for all β-lactam drugs (penicillins, cepha-
losporins, and carbapenems). As a result, staphylococci that possess
this gene are also resistant to the penicillinase-resistant penicillins
(oxacillin and methicillin), and so are termed methicillin-resistant
Staphylococcus (MRS) species. Many MRS also possess resistance
genes to other antimicrobial drug classes. There is no evidence that
MRS are more virulent than methicillin-susceptible staphylococci,
but infections with MRS are more difficult to treat with antimicro-
bial drugs. The prevalence of MRS isolated from dogs has ranged
from 0.6% to as high as 67%, depending on the population sam-
pled (healthy versus disease, hospitalized versus outpatients), geo-
graphic location, study dates, and sites of specimen collection. Of
89 staphylococcal isolates from dogs with superficial pyoderma
seen in 2010 and 2011 by the University of California, Davis, Vet-
erinary Dermatology service, 38.2% were MRS. This compared
with 27.3% of 33 isolates from a primary care clinic in the same
region.5 Methicillin-resistant S. pseudintermedius (MRSP) isolates
are generally resistant to more antimicrobial drug classes than
methicillin-resistant S. aureus (MRSA) isolates from dogs and cats.
Severe multidrug resistance is also prevalent among CoNS.
Staphylococcus pseudintermedius
S. pseudintermedius is the most common Staphylococcus species
isolated from dogs. It is a commensal of the skin and mucous
membranes of dogs and cats and possibly one of the most com-
mon bacterial pathogens treated by veterinarians. It primar-
ily colonizes the anal mucosa and nares of healthy dogs and
cats, but it can also be isolated from the mouth, forehead, and
347
348 SECTION 2  Bacterial Diseases
A genesis of infections caused by this organism.8 Like S. aureus,
B antimicrobial drugs. Risk factors for MRSP infection identified
FIGURE 35-1  Histopathology of the integument from the left inguinal region and
flank of an 8-year old male neutered boxer dog that had severe, regionally extensive acute
neutrophilic cellulitis and panniculitis accompanied by septic shock. A methicillin suscep-
tible Staphylococcus pseudintermedius was cultured from an aspirate of cellulitis fluid.
The dog had also been treated with prednisone and azathioprine for 4 years to maintain
remission for immune-mediated hemolytic anemia and thrombocytopenia. A, H&E stain.
Large numbers of degenerate neutrophils are present. B, Staining with Brown and Benn
stain reveals numerous of gram-positive coccoid bacteria, individually, in pairs, and in clus-
ters.1000× oil magnification.
inguinal region. It can colonize the skin transiently following
grooming and licking in dogs with pruritis. Cats have a much
lower prevalence of colonization with S. pseudintermedius than
dogs. S. pseudintermedius invades tissues opportunistically
and is a major cause of superficial and deep pyoderma, otitis
externa, urinary tract infections (UTIs), and wound and surgical
site infections, as well as bronchopneumonia, ocular infections,
bacteremia, osteomyelitis, and infections of body cavities. An
underlying immunosuppressive condition or break in host bar-
riers is present in the majority of infections.
Before 2005, S. pseudintermedius were identified as S. inter-
medius. The name S. pseudintermedius was defined when isolates
from a dog, cat, horse, and parrot were analyzed using molecu-
lar methods.6 Subsequently, a molecular analysis of organisms
from dogs and cats that had been previously identified as S. inter-
medius revealed that they were actually S. pseudintermedius.
S. pseudintermedius belongs to the “S. intermedius group” (SIG),
which comprises the three closely related species S. intermedius,
S. pseudintermedius, and S. delphini. It is difficult to differenti-
ate among these species with phenotypic methods alone; genetic
typing methods are required. Because genetic typing methods are
not available for routine identification in veterinary diagnostic
laboratories, isolates from dogs with phenotypic characteristics
typical of S. intermedius are now generally identified as S. pseud-
intermedius.7 However, some laboratories still describe strains
from dogs that have biochemical characteristics that do not quite
match that typical for the type strain of S. pseudintermedius as
“SIG organisms.”
The whole genome of S. pseudintermedius has been sequenced,
which is a major contribution to our understanding of the patho-
S. pseudintermedius possesses numerous mobile genetic elements
and potential virulence factors, such as toxin genes that encode
for a superantigen, leukotoxin, hemolysins, and exfoliative tox-
ins, as well as an array of proteases.
Methicillin resistance has emerged as a serious and wide-
spread problem in S. pseudintermedius isolates from both
healthy and sick dogs and cats. Molecular analysis has shown
that among dogs, two distinct, major clones of MRSP have
spread across Europe and North America, respectively.9 Many
isolates of S. pseudintermedius not only are methicillin resistant,
but also acquire resistance genes to other classes of antimicrobi-
als, including tetracyclines, trimethoprim-sulfonamides, fluoro-
quinolones, chloramphenicol, and macrolides. Isolates that are
resistant to three or more different classes of antimicrobial drugs
are defined as multiple drug resistant (MDR) isolates. Currently,
many isolates remain susceptible to chloramphenicol and amino-
glycosides, but the prevalence of resistance to chloramphenicol
has increased among MRSP isolates. MDR S. pseudintermedius
isolates are no more virulent or difficult to disinfect than sus-
ceptible isolates, but they are more difficult to eliminate using
in dogs include previous hospitalization, living in an urban envi-
ronment, older age, and, most consistently, previous antimicro-
bial drug treatment. Household contacts of dogs and cats with
MRSP infection are also frequently colonized with MRSP.10
Staphylococcus aureus
S. aureus colonizes the skin and mucous membranes of humans
and is one of the most common community-acquired and
­hospital-acquired bacterial pathogens of people. The anterior
nares and the throat are preferred niches for the organism,
which can be isolated from the anterior nares of up to 30%
of the healthy human population. Methicillin resistance is a
growing problem among isolates from humans and animals.
Both hospital-acquired methicillin-resistant (HA-MRSA) and
community-acquired methicillin-resistant (CA-MRSA) strains
have been identified in infected human patients, and invasive
S. aureus infections are associated with high mortality rates.
When compared with S. pseudintermedius, S. aureus tends
not to colonize dogs. For example, staphylococci were isolated
from 37 of 50 healthy dogs from Pennsylvania, and 6 (12%) of

the dogs were colonized with S. aureus; 68% were colonized with
S. pseudintermedius.11 MRSA strains that colonize dogs may be
identical to those that infect in-contact humans, but sometimes
strains found on dogs differ from those found on in-contact
humans.12 The chance that a dog that is in contact with an
infected human will be colonized with MRSA decreases with the
time after diagnosis of human MRSA infection, which suggests
transient carriage of MRSA by dogs. MRSA was not transferred
between apparently healthy dogs in a kennel of dogs, and after
treatment of an MRSA wound infection in one dog, all dogs tested
negative for MRSA within 2 weeks.13 Although staphylococci
are isolated less commonly from cats, colonization with S. aureus
is more prevalent and certain S. aureus strains may be commen-
sals of cats; staphylococci were isolated from 17 of 50 healthy
cats, 59% of which were colonized with S. aureus and 65% with
S. pseudintermedius.14
In dogs and cats, S. aureus infection has been associated with
clinical manifestations that include pyoderma, otitis externa, UTIs,
surgical site infections, osteomyelitis, and bacteremia. Although
MRSA infections are uncommonly diagnosed in dogs and cats,
they have been increasingly recognized over the past 2 decades.
When canine infections with MRSA do occur, they are often of
human hospital origin, but human community-acquired strains
have also been isolated. In one study, strong risk factors for MRSA
infection (as opposed to methicillin-susceptible S. aureus [MSSA]
infection) were the number of antimicrobial courses administered,
the number of days admitted to veterinary clinics, and a history
of surgical implant placement.15 There has been concern that the
use of certain antibiotics, such as cephalosporins and fluoroquino-
lones, may select for MRSA infections in dogs.
Staphylococcus schleiferi
Coagulase-positive and coagulase-negative S. schleiferi have
primarily been isolated from dogs with superficial pyoderma
and otitis externa, as well as from the ear canals of healthy
dogs. Rarely, UTIs and respiratory infections with S. schleiferi
have been described in dogs.3 In dogs with pyoderma and otitis
externa, S. schleiferi is not as prevalent as S. pseudintermedius
but in most studies has been isolated as often, or more often,
than S. aureus. Coagulase-negative S. schleiferi is a commensal
of the human axilla but can cause disease in immunosuppressed
humans. Coagulase-positive S. schleiferi may be specialized to
colonize dogs.
S. schleiferi isolates from dogs with skin disease are often
methicillin-resistant as a result of mecA gene carriage. In a
study of 225 S. schleiferi isolates from dogs from the eastern
United States, of which 52% were coagulase negative and 42%
were coagulase positive, methicillin resistance was identified in
57% of the isolates.3 Methicillin resistance was more prevalent
among coagulase-negative isolates. Isolates from dogs with pyo-
derma may be more likely to be methicillin resistant than those
from dogs with otitis externa.
Clinical Features
Signs and Their Pathophysiology
Staphylococci can cause pyoderma, otitis externa, pneumonia,
UTIs, soft tissue infections, surgical site infections, bacteremia,
ocular infections, and endocarditis in dogs and cats.
S. aureus and S. pseudintermedius possess several adhes-
ins and produce an array of toxins. The clinical effect of these
toxins is best characterized for S. aureus infections in humans.
CHAPTER 35  Staphylococcus Infections 349
A number of enterotoxins have been identified in S. aureus and
S. pseudintermedius; these can cause food poisoning in humans
but have not been recognized as a cause of disease in dogs and
cats. Staphylococcal enterotoxins are heat stable, so they are not
inactivated by cooking, even when the bacteria themselves are
destroyed. Staphylococcal toxic shock syndrome results from
the expression of superantigens, which include toxic shock syn-
drome toxin 1, as well as a large number of enterotoxins and
exotoxins that can also act as superantigens. Cellulitis, necrotiz-
ing fasciitis, and toxic shock syndrome has been rarely reported
in dogs infected with S. pseudintermedius.16,17 Most toxic shock
syndrome and necrotizing fasciitis cases in dogs and cats are
instead caused by Streptococcus canis (see Chapter 34). Scalded
skin syndrome results from infection of humans with S. aureus
strains that produce an exfoliative toxin, which hydrolyses the
intercellular glycoprotein desmoglein-1. Strains of S. pseudin-
termedius that possess exfoliative toxin genes have been isolated
more commonly from dogs with pyoderma than from healthy
dogs, so exfoliative toxin may also play a role in canine disease.
Panton-Valentine toxin (PVL) is a leukocidin that is found in
CA-MRSA strains but only a small percentage of HA-MRSA
and MSSA strains, and strains that encode PVL have been asso-
ciated with skin and soft tissue infections and hemorrhagic
pneumonia in children and young adults. Although a similar
toxin has been identified in S. pseudintermedius, the role of PVL
in the pathogenesis of staphylococcal infections in dogs and cats
is unknown.
Some strains of S. pseudintermedius and S. aureus produce
biofilm, which may contribute to the ability of these organisms
to persist in the environment, cause nosocomial infections, and
chronic recurrent infections that respond poorly to antimicro-
bial treatment.18
Diagnosis
Laboratory Abnormalities
Complete Blood Count, Biochemistry, and Urinalysis Findings
Dogs with staphylococcal infections of the skin and ear canal
often have no clinically significant hematologic or biochemical
findings. Systemic infections may be associated with neutrophilia
and a left shift, toxic neutrophils, lymphopenia, and monocyto-
sis. The urinalysis of dogs and cats with staphylococcal UTIs
may reveal pyuria, proteinuria, or hematuria, and sometimes
cocci in clusters may be evident on sediment examination.
Cytologic Abnormalities
Aspirates of affected tissues or pus or respiratory lavage fluid
specimens from animals with staphylococcal infections may
contain large numbers of degenerate and nondegenerate neutro-
phils. The presence of cocci in clusters suggests staphylococcal
infection.
Microbiological Tests
Isolation and Identification
Staphylococci that infect dogs and cats are readily isolated on
routine bacteriologic media from aspirates, tissue biopsies, body
fluid, and lavage specimens. Colonies typically appear within
24 hours. Staphylococcal species are identified on the basis
of phenotypic and biochemical reactions such as production
of coagulase and/or clumping factor, production of hemoly-
sins, ability to ferment carbohydrates, and acetoin production
(Vogues-Proskauer reaction). PCR and sequencing of ribosomal
350 SECTION 2  Bacterial Diseases
RNA genes (16S or 23S) may be necessary for identification
of some species. CoNS are often not identified to the species
level because molecular techniques are often required for accu-
rate identification. Because CoNS are of low pathogenicity,
they may be contaminants, especially if they are isolated from
nonsterile sites, growth is minimal (1+), or another pathogen
is present. However, they may occasionally be pathogenic. In
particular, isolation of S. felis from urine or coagulase-negative
S. schleiferi from dogs with pyoderma or otitis may have clinical
significance.
Evaluation of antimicrobial susceptibility is of critical impor-
tance because of the high prevalence of methicillin-resistant and
multidrug-resistant staphylococci in some parts of the world.
Susceptibility testing for staphylococci should be performed
according to recommendations of groups such as the Clini-
cal and Laboratory Standards Institute (CLSI). Oxacillin and
cefoxitin are used in the laboratory as a surrogate for detection
of methicillin resistance. Laboratories should report methicillin-
resistant staphylococci as resistant to all other penicillins, car-
bapenems, cephalosporins, and β-lactam/β-lactamase inhibitor
combinations, regardless of the results of in vitro susceptibility
test results for these agents.
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR assays have been developed that detect and dif-
ferentiate some Staphylococcus species, as well as identify the
presence of the mecA gene. These assays are available in some
laboratories for diagnosis of MRSA infections in humans, but
to date have only been used on a research basis to characterize
staphylococci isolated from dogs and cats.
Treatment and Prognosis
The approach to treatment of staphylococcal infections depends
on the site of infection and the severity of disease; the reader is
TABLE 35-1
Suggested Antimicrobial Drugs for Treatment of Staphylococcal Infections in Dogs and Cats
Predicted/Determined
Staphylococcal
Susceptibility Drug
Methicillin-susceptible Ampicillin sodium
Cefazolin sodium
Cephalexin
Methicillin-resistant* Trimethoprim-sulfamethoxazole
Clindamycin†
Doxycycline
Rifampin
Amikacin
Vancomycin‡
Linezolid‡
Nitrofurantoin (macrocrystalline
formulation)§
*Susceptibility testing recommended if methicillin resistance is likely based on regional or hospital prevalence data. Many isolates are resistant to
clindamycin, trimethoprim-sulfamethoxazole, doxycycline; some are resistant to chloramphenicol. Consider topical antibacterial treatment if possible.
†May not be effective if erythromycin resistance is present on the susceptibility panel due to inducible clindamycin resistance.
‡Reserve for serious infections that are resistant to all other reasonable alternatives, on the basis of culture and susceptibility testing. See Table 8-6
for vancomycin administration protocol.
§For resistant urinary tract infections only.
referred to Chapters 84 to 90 for treatment recommendations
for different organ systems. Identification and management
of the underlying cause (rather than repeated treatment with
antimicrobial drugs) is of paramount importance to minimize
selection for resistant organisms. Any foreign material (such as
catheters, implants) must be removed. Bathing and topical treat-
ments should be considered for pyoderma as an alternative to
antimicrobial drug therapy (see Chapter 84). The use of topi-
cal wound treatments such as accelerated hydrogen peroxide,
chlorhexidine, or silver sulfadiazine could also be considered
provided infection is superficial.
When systemic antimicrobial treatment is necessary, staphy-
lococcal infections should ideally be treated based on the results
of culture and susceptibility testing and/or knowledge of the
regional prevalence of methicillin resistance and multidrug resis-
tance, because this can vary, not only between countries but also
between different states or regions within a state. In regions where
methicillin resistance is rare, a β-lactam drug such as cephalexin,
clavulanic acid–amoxicillin, or a penicillinase-resistant penicillin
could be used empirically (Table 35-1), and culture and suscepti-
bility testing may not be necessary for uncomplicated infections
such as newly diagnosed pyoderma or UTIs.
Where methicillin resistance is prevalent in the region, culture
and susceptibility testing is recommended. If this is not possible,
a reasonable first choice for uncomplicated infections that are
not life threatening is clindamycin. Clindamycin is an attractive
choice given that many infections are skin, orthopedic, or soft
tissue infections (sites to which clindamycin distributes well);
it is inexpensive, inhibits toxin production by staphylococci,
and is available in oral and intravenous formulations. However,
some MRS demonstrate inducible clindamycin resistance, a
phenomenon whereby the organism is susceptible to clindamy-
cin in vitro but rapidly develops resistance to clindamycin when
exposed to macrolides (such as erythromycin) or lincosamides
in vivo, owing to bacterial methylation of the ribosomal binding
Dose (mg/kg) Interval (hours) Route
10-20 6-8 IV, IM, SC
20-35 8 IV, IM
10-30 (dogs), 15-20 (cats) 12 PO
30 12 PO, IV
11 12 PO
10 12 PO
5-10 12 PO
15-30 (dogs), 10-14 (cats) 24 IV, IM, SC
15 6-8 (dogs), 8 (cats) IV
10 (dogs) 8 PO, IV
2-3 8 PO

site for clindamycin.19 The end result is treatment failure or
relapse, although some treatment successes have been reported
in human patients infected with S. aureus strains that have
inducible clindamycin resistance. Staphylococci with inducible
clindamycin resistance are resistant to macrolides such as eryth-
romycin and susceptible to clindamycin in routine susceptibil-
ity tests. Nevertheless, not all staphylococci with this resistance
pattern demonstrate inducible clindamycin resistance. Identifi-
cation of inducible clindamycin resistance requires a special test
known as a D-zone test. It involves culture of the organism on
agar in the presence of an erythromycin and clindamycin disk.
Organisms near to the erythromycin disk express enhanced
resistance to clindamycin, which results in a D shape to the
zone of inhibition around the clindamycin disk (Figure 35-2).
At the time of writing, inducible clindamycin resistance appears
to be prevalent in S. aureus isolates from dogs and cats (18%
in one study of 62 isolates) but is rarely reported in S. pseud-
intermedius (0 of 46 isolates in one study, and 1 of 60 isolates
in another study).20,21 As a result, it is reasonable to assume
that disease caused by S. pseudintermedius isolates that are sus-
ceptible to clindamycin on routine susceptibility testing is likely
to respond to treatment with clindamycin. However, in some
geographic locations, a high percentage of canine MRSP isolates
are overtly resistant to clindamycin, which suggests that clinda-
mycin may not be an ideal empiric treatment choice in these
locations. The use of fluoroquinolones to treat staphylococcal
infections has been controversial, because they have been asso-
ciated with treatment failures and development of methicillin
resistance in human S. aureus infections. In one study, a fluoro-
quinolone control program in a tertiary human hospital led to a
significant reduction in fluoroquinolone-resistant Pseudomonas
aeruginosa and MRSA rates over 4 years.22
If severe and invasive infection with an MRS is a possibil-
ity, a combination of clindamycin and an aminoglycoside
could be administered parenterally, pending the results of cul-
ture and susceptibility testing. Antimicrobials that could be
considered for life-threatening methicillin-resistant and multi-
drug-resistant staphylococcal infections include trimethoprim-
sulfamethoxazole, doxycycline, aminoglycosides, rifampin,
chloramphenicol, vancomycin, tigecycline, quinupristin-dal-
fopristin, and linezolid (see Chapter 8). Some MRS are also
FIGURE 35-2  Positive D-zone test for inducible clindamycin resistance in staphylo-
cocci. Organisms exposed to erythromycin express enhanced resistance to clindamycin,
which leads to distortion of the zone of inhibition around the clindamycin disk. (Courtesy
Dr. Scott Weese, University of Guelph.)
CHAPTER 35  Staphylococcus Infections 351
resistant to trimethoprim-sulfamethoxazole, doxycycline, and
chloramphenicol, and toxicity may limit the usefulness of trim-
ethoprim-sulfamethoxazole and chloramphenicol (see Chapter
8). Aminoglycosides must be given parenterally and have the
potential to cause nephrotoxicity, and their activity is reduced
in the presence of pus. The use of rifampin alone is contro-
versial, because rifampin monotherapy has been associated
with development of resistance in S. aureus infections.23 As a
result, the use of rifampin in combination with other drugs has
been recommended. Minocycline has received attention as an
alternative to doxycycline because some doxycycline-resistant
strains (strains that possess the tetracycline efflux protein tetK)
remain susceptible to minocycline. In contrast, strains that pos-
sess tetM (which protects the ribosome from tetracycline bind-
ing) are resistant to all tetracyclines. Additional information on
the pharmacokinetics and definition of clinical breakpoints for
minocycline are required.
Vancomycin, tigecycline, daptomycin, quinupristin-
dalfopristin, and linezolid are important drugs for MRSA
infections in humans. Whether these drugs should be used to
treat companion animals has been debated, given that they are
already widely used in humans and that they may be required
to save the lives of companion animals. Cost and/or the need
for parenteral administration (vancomycin, tigecycline, dapto-
mycin, and quinupristin-dalfopristin) can also limit the useful-
ness of these drugs in companion animals. At a minimum, their
use should be reserved for situations when no other treatment
alternatives exist and when the infection is life threatening but
has the potential to be resolved with appropriate treatment. The
use of vancomycin or linezolid may be indicated in dogs with
bacteremia or endocarditis due to MRSA, MRSP, or methicillin-
resistant CoNS that are resistant to all options other than amino-
glycosides and concerns for aminoglycoside toxicity exist.
Prevention
Prevention of staphylococcal infections involves attention to
the underlying cause of disease (such as allergic dermatitis), and
strict veterinary clinic and veterinary hospital infection control
practices (see Chapter 11). Because canine MRSA infections
may be acquired from colonized or infected humans, trans-
mission to dogs may be reduced by hand washing and general
surface cleaning and disinfection in the home environment (see
Public Health Aspects, next).
Public Health Aspects
Most concern regarding the public health risk of staphylococcal
infection or colonization in companion animals has related to
MRSA. However, because colonization of dogs with MRSA is
transient, other humans are a more significant source of human
MRSA infection. Nevertheless, some studies suggest that veteri-
narians who work with small animals may be at increased risk for
colonization with MRSA. Although around 30% of the human
population is colonized with S. aureus, the prevalence of MRSA
colonization is generally much lower than this, less than 2%. In
Australia, dog and cat veterinarians had a fivefold higher risk for
MRSA carriage when compared with industry and government
veterinarians.24 Small animal veterinarians in the United King-
dom were also at increased risk of colonization.25 In a study from
the United States, 17.3% of veterinarians and veterinary techni-
cians were colonized, and the rate of colonization did not differ
352 SECTION 2  Bacterial Diseases
between small- and large-animal veterinarians.26 In another study,
only large-animal practice was associated with colonization.27
Regardless of the direction of transmission (human to pet
versus pet to human), owners of dogs and cats diagnosed with
MRSA infection should be educated about the need to wash
their hands after handling their pets, clean and disinfect surfaces
that might become contaminated, and not let their pet lick their
face or open wounds. Infected sites should be covered with a
suitable dressing to minimize contamination of the environment
whenever possible. Owners should be instructed to talk to their
health care providers if they have concerns about their health or
the health of others in their household. In the hospital environ-
ment, transmission of MRSA may be prevented through the use
of a strict hospital infection control program (see Chapter 11).
CASE EXAMPLE
Signalment: “Elliot,” a 10-year-old male neutered miniature
schnauzer from San Francisco, CA
History: Elliot was seen for a possible pacemaker infection.
One month after placement of a dual-chamber pacemaker
for sick sinus syndrome, a fluctuant mass was noted
around the generator. The seroma was aspirated, and
cytologic examination of the aspirate showed many
neutrophils. Treatment with clavulanic acid-amoxicillin
was initiated (25 mg/kg PO q12h). One month later, there
had been no apparent improvement. The wound was
flushed with chlorhexidine solution and treatment with
chloramphenicol was initiated, but the problem persisted.
The chloramphenicol was discontinued, and the dog was
referred for further evaluation. The owners reported no
vomiting, diarrhea, or decreased appetite. Elliot had also
been diagnosed with early myxomatous degeneration of
the mitral valve based on the results of echocardiography,
associated with a grade III/VI left apical systolic murmur at
the time of pacemaker placement.
Physical Examination:
Body Weight: 9.8 kg
General: Quiet, alert, and responsive. Slightly tacky but pink
mucous membranes, capillary refill time <2 s, approximately
5% dehydrated, T = 104°F, HR = 160 beats/min, RR = 40
breaths/min.
Eyes, Ears, Nose, and Throat: A mild crusted ocular discharge
was present bilaterally, as well as mild serous nasal discharge,
and moderate periodontal disease.
Integument: A 1-cm wound that discharged purulent material
was present in the region of the right jugular incision, with a
draining tract located along the jugular lead that extended
to the pacemaker generator over the right cervical region.
There was an accumulation of fluid that measured 1 cm in
diameter over the generator.
Musculoskeletal: Ambulatory with normal gait, BCS 5/9
Cardiovascular: Heart rate 160 beats/min, regular rhythm,
grade III/VI left apical systolic murmur, femoral pulses strong
and synchronous
Respiratory: Mild crackles were auscultated in the left and
right caudal lung fields.
Abdominal Palpation: Cranial organomegaly was present.
Routine screening and decontamination is not recommended
because dogs appear to resolve infection over time. Topical
mupirocin, which has been used for decontamination in humans,
is not likely to be effective in dogs and cats given the large size of
their nasal cavities, and concerns have been raised regarding the
need for and efficacy of decolonization with systemic antibiot-
ics, which may only select for antimicrobial resistance.
Human infections with S. pseudintermedius occur occasion-
ally in association with pet contact, although the organism
generally tends not to colonize humans. However, coloniza-
tion with MRSP was reported more frequently in small-animal
dermatologists than colonization with MRSA.28,29 Precautions
recommended for MRSA infection are also indicated for MRSP
infections.
Lymph Nodes: All lymph nodes were normal in size.
Laboratory Findings:
CBC:
HCT 32.2% (40%-55%)
MCV 60.3 fL (65-75 fL)
MCHC 32.3 g/dL (33-36 g/dL)
Reticulocytes 9100 cells/µL (7000-65,000 cells/µL)
WBC 29,680 cells/µL (6000-13,000 cells/µL)
Neutrophils 22,854 cells/µL (3000-10,500 cells/µL)
Lymphocytes 4155 cells/µL (1000-4000 cells/µL)
Monocytes 594 cells/µL (150-1200 cells/µL)
Eosinophils 890 cells/µL (0-1500 cells/µL)
Platelets 446,000 platelets/µL (150,000-400,000 platelets/µL)
Slight toxicity of neutrophils and band neutrophils.
Serum Chemistry Profile:
Sodium 140 mmol/L (145-154 mmol/L)
Potassium 5.6 mmol/L (3.6-5.3 mmol/L)
Chloride 103 mmol/L (108-118 mmol/L)
Bicarbonate 16 mmol/L (16-26 mmol/L)
Phosphorus 7.5 mg/dL (3.0-6.2 mg/dL)
Calcium 11.3 mg/dL (9.7-11.5 mg/dl)
BUN 48 mg/dL (5-21 mg/dL)
Creatinine 1.8 mg/dL (0.3-1.2 mg/dL)
Glucose 81 mg/dL (64-123 mg/dL)
Total protein 7.6 g/dL (5.4-7.6 g/dL)
Albumin 2.7 g/dL (3.0-4.4 g/dL)
Globulin 4.9 g/dL (1.8-3.9 g/dL)
ALT 61 U/L (19-67 U/L)
AST 17 U/L (19-42 U/L)
ALP 190 U/L (21-170 U/L)
Creatine kinase 61 U/L (51-399 U/L)
Gamma GT 3 U/L (0-6 U/L)
Cholesterol 323 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL)
Magnesium 1.7 mg/dL (1.5-2.6 mg/dL).
Urinalysis: SGr 1.013; pH 6.0, 150 mg/dL protein, no bilirubin,
150 erythrocytes/µL hemoprotein, no glucose, no ketones,
12-18 WBC/HPF, 8-12 RBC/HPF, no other significant findings.
Coagulation Panel: PT 8.7 s (7.0-9.3 s), APTT 14.1 s (10.4-12.9 s),
fibrinogen 412 mg/dL (109-311 mg/dL), D-dimers 418 ng/
mL (0-186 ng/mL), antithrombin 84% (80%-120%).
Imaging Findings:
Thoracic Radiographs: See Figure 35-3. Mild cardiomegaly
was present. The far caudal left pulmonary vasculature

FIGURE 35-3  Dorsoventral thoracic radiograph from a 10-year-old male neu-
tered miniature schnauzer with methicillin-resistant Staphylococcus bacteremia and
pacemaker infection. There was a bronchointerstitial pattern and mild cardiomegaly.
The caudal left pulmonary vasculature was indistinct with patchy infiltrates (arrows),
which raised suspicion for septic embolic pneumonia.
was indistinct with region of patchy infiltrates (arrows).
The pacemaker was static in appearance with a generator
embedded in the tissues dorsal to the cervical spine and two
leads extending into the region of the cardiac silhouette—
one terminating at the level of the right atrium and the
other terminating at the level of the right ventricle. Within
the viewable abdomen, the liver was persistently markedly
enlarged and rounded. Given the absence of vasculature in
the left caudal lung lobe, embolism in this area was suspected.
Echocardiogram (to evaluate for endocarditis): Mild mitral
regurgitation was present, with no evidence of endocarditis.
Microbiologic Testing:
Aerobic Blood Culture and Susceptibility: Bottle 1 (right
cephalic catheter): Direct smear from bottle: small numbers
of gram-positive cocci and small numbers of gram-variable
rods. Culture: coagulase-negative Staphylococcus and
Serratia marcescens. The CoNS was methicillin resistant
and resistant to clindamycin, enrofloxacin, marbofloxacin,
erythromycin, trimethoprim-sulfamethoxazole; had
intermediate susceptibility to gentamicin (8.00 µg/
mL); and was susceptible to amikacin (≤4.00 µg/mL),
chloramphenicol (8.00 µg/mL), doxycycline (4.00 µg/mL),
rifampin (≤1 µg/mL), daptomycin (≤0.25 µg/mL), linezolid
CHAPTER 35  Staphylococcus Infections 353
(≤0.5 µg/mL), and vancomycin (≤1.00 µg/mL). The Serratia
marcescens was resistant to cefazolin, cefoxitin, cephalothin,
and doxycycline; and susceptible to amikacin (≤4.00 µg/
mL), amoxicillin-clavulanic acid (4.00 µg/mL), ampicillin
(≤2.00 µg/mL), cefovecin (1.00 µg/mL), cefpodoxime (≤2.00
µg/mL), enrofloxacin (≤0.25 µg/mL), marbofloxacin (≤1
µg/mL), gentamicin (≤1 µg/mL), imipenem (≤1 µg/mL),
ticarcillin (≤8 µg/mL), ticarcillin-clavulanate (≤8 µg/mL), and
trimethoprim-sulfamethoxazole (≤0.5 µg/mL).
Bottle 2 (right lateral saphenous vein): Direct smear: small num-
bers of gram-positive cocci. Culture: coagulase-negative
Staphylococcus.
Urine aerobic culture and susceptibility (cystocentesis
specimen): >105 organisms/mL coagulase-negative
Staphylococcus, with an identical antibiogram to that of the
isolate from the blood.
Seroma Aerobic and Anaerobic Culture and Susceptibility:
Small numbers of coagulase-negative Staphylococcus, with
an identical antibiogram to that of the isolate from the
blood and urine.
Diagnosis: Bacteremia and probable embolic pneumonia
due to methicillin-resistant Staphylococcus spp. and possibly
Serratia marcescens associated with pacemaker placement.
Skin/soft tissue and urinary tract infection with methicillin-
resistant Staphylococcus spp.
Treatment: Elliot was hospitalized with full-contact
precautions that included the use of gloves and gowns and
contact with a limited number of individuals. Treatment was
initiated with intravenous fluids, vancomycin (10 mg/kg IV
q6h) for the methicillin-resistant staphylococcal infection,
and enrofloxacin (10 mg/kg IV q24h) for a possible Serratia
marcescens infection. After 48 hours of treatment, the
pacemaker was removed and replaced with a ventricular
(VVIR) epicardial pacemaker, and a Penrose drain was
placed at the incision site. A repeat blood culture (four
bottles) performed 3 days later showed growth of the
CoNS in one bottle. The pulmonary infiltrates resolved,
creatinine normalized, and repeat blood and urine cultures
were negative after 10 days of vancomycin treatment.
The dog was discharged from the hospital. Follow-up
cultures after an additional 10 days off antibiotics remained
negative. Six months later, Elliot developed right-sided
congestive heart failure with ascites and pleural effusion.
Aerobic bacterial culture of the ascites fluid was negative.
On echocardiography, the tricuspid valve was severely
thickened and irregular. Endocarditis was suspected.
Additional diagnostics that included blood cultures were
declined by the owners, who elected euthanasia without
necropsy.
Comments: Vancomycin was used to treat this severe
methicillin-resistant staphylococcal infection because
the dog had renal failure and there was concern for
aminoglycoside toxicity. The S. marcescens may have
represented a contaminant, but it was treated because of
the severity of the dog’s condition. Whether the endocarditis
that developed was the result of a new infection or
persistence of the original staphylococcal infection could
not be determined.
354 SECTION 2  Bacterial Diseases
SUGGESTED READING
Papich MG. Selection of antibiotics for meticillin-resistant Staphylococ-
cus pseudintermedius: time to revisit some old drugs? Vet Dermatol.
2012;23(4):352-360.
REFERENCES
1. Ogsten A. Micrococcus poisoning. J Anat Physiol. 1882;16:526.
2. Ghebremedhin B, Layer F, Konig W, et  al. Genetic classification
and distinguishing of Staphylococcus species based on different
partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences.
J Clin Microbiol. 2008;46:1019-1025.
3. Cain CL, Morris DO, O’Shea K, et  al. Genotypic relatedness and
phenotypic characterization of Staphylococcus schleiferi subspecies in
clinical samples from dogs. Am J Vet Res. 2011;72:96-102.
4. Litster A, Moss SM, Honnery M, et al. Prevalence of bacterial spe-
cies in cats with clinical signs of lower urinary tract disease: rec-
ognition of Staphylococcus felis as a possible feline urinary tract
pathogen. Vet Microbiol. 2007;121:182-188.
5. Eckholm NG, Outerbridge CA, White SD, et al. Prevalence of and
risk factors for infection with methicillin-resistant Staphylococcus
spp. in dogs with pyoderma in northern California. Submitted.
2012.
6. Devriese LA, Vancanneyt M, Baele M, et al. Staphylococcus pseud-
intermedius sp. nov., a coagulase-positive species from animals. Int
J Syst Evol Microbiol. 2005;55:1569-1573.
7. Devriese LA, Hermans K, Baele M, et  al. Staphylococcus pseud-
intermedius versus Staphylococcus intermedius. Vet Microbiol.
2009;133:206-207.
8. Ben Zakour NL, Bannoehr J, van den Broek AH, et al. Complete
genome sequence of the canine pathogen Staphylococcus pseudin-
termedius. J Bacteriol. 2011;193:2363-2364.
9. Perreten V, Kadlec K, Schwarz S, et al. Clonal spread of methicillin-
resistant Staphylococcus pseudintermedius in Europe and North
America: an international multicentre study. J Antimicrob Che-
mother. 2010;65:1145-1154.
10. van Duijkeren E, Kamphuis M, van der Mije IC, et al. Transmis-
sion of methicillin-resistant Staphylococcus pseudintermedius
between infected dogs and cats and contact pets, humans and the
environment in households and veterinary clinics. Vet Microbiol.
2011;150:338-343.
11. Griffeth GC, Morris DO, Abraham JL, et al. Screening for skin car-
riage of methicillin-resistant coagulase-positive staphylococci and
Staphylococcus schleiferi in dogs with healthy and inflamed skin.
Vet Dermatol. 2008;19:142-149.
12. Morris DO, Lautenbach E, Zaoutis T, et al. Potential for pet ani-
mals to harbour methicillin-resistant Staphylococcus aureus when
residing with human MRSA patients. Zoonoses Public Health.
2012;59(4):286-293.
13. Loeffler A, Pfeiffer DU, Lindsay JA, et  al. Lack of transmission
of methicillin-resistant Staphylococcus aureus (MRSA) between
apparently healthy dogs in a rescue kennel. Vet Microbiol.
2010;141:178-181.
14. Abraham JL, Morris DO, Griffeth GC, et al. Surveillance of healthy
cats and cats with inflammatory skin disease for colonization of the skin
by methicillin-resistant coagulase-positive staphylococci and Staphy-
lococcus schleiferi ssp. schleiferi. Vet Dermatol. 2007;18:252-259.
15. Soares Magalhaes RJ, Loeffler A, Lindsay J, et al. Risk factors for
methicillin-resistant Staphylococcus aureus (MRSA) infection in
dogs and cats: a case-control study. Vet Res. 2010;41:55.
16. Girard C, Higgins R. Staphylococcus intermedius cellulitis and
toxic shock in a dog. Can Vet J. 1999;40:501-502.
17. Weese JS, Poma R, James F, et al. Staphylococcus pseudintermedius
necrotizing fasciitis in a dog. Can Vet J. 2009;50:655-656.
18. Osland AM, Vestby LK, Fanuelsen H, et  al. Clonal diversity
and biofilm-forming ability of methicillin-resistant Staphylococ-
cus pseudintermedius. J Antimicrob Chemother. 2012 Apr, 67:
841-848.
19. Lewis 2nd JS, Jorgensen JH. Inducible clindamycin resistance in
staphylococci: should clinicians and microbiologists be concerned?
Clin Infect Dis. 2005;40:280-285.
20. Rubin JE, Ball KR, Chirino-Trejo M. Antimicrobial susceptibility
of Staphylococcus aureus and Staphylococcus pseudintermedius
isolated from various animals. Can Vet J. 2011;52:153-157.
21. Faires MC, Gard S, Aucoin D, et al. Inducible clindamycin-resistance
in methicillin-resistant Staphylococcus aureus and methicillin-
resistant Staphylococcus pseudintermedius isolates from dogs and
cats. Vet Microbiol. 2009;139:419-420.
22. Lafaurie M, Porcher R, Donay JL, et  al. Reduction of fluoroqui-
nolone use is associated with a decrease in methicillin-resistant
Staphylococcus aureus and fluoroquinolone-resistant Pseudomo-
nas aeruginosa isolation rates: a 10 year study. J Antimicrob Che-
mother. 2012;67(4):1010-1015.
23. Falagas ME, Bliziotis IA, Fragoulis KN. Oral rifampin for eradica-
tion of Staphylococcus aureus carriage from healthy and sick popu-
lations: a systematic review of the evidence from comparative trials.
Am J Infect Control. 2007;35:106-114.
24. Jordan D, Simon J, Fury S, et al. Carriage of methicillin-resistant
Staphylococcus aureus by veterinarians in Australia. Aust Vet J.
2011;89:152-159.
25. Loeffler A, Pfeiffer DU, Lloyd DH, et al. Meticillin-resistant Staph-
ylococcus aureus carriage in UK veterinary staff and owners of
infected pets: new risk groups. J Hosp Infect. 2010;74:282-288.
26. Burstiner LC, Faires M, Weese JS. Methicillin-resistant Staphylo-
coccus aureus colonization in personnel attending a veterinary sur-
gery conference. Vet Surg. 2010;39:150-157.
27. Hanselman BA, Kruth SA, Rousseau J, et al. Methicillin-resistant
Staphylococcus aureus colonization in veterinary personnel. Emerg
Infect Dis. 2006;12:1933-1938.
28. Paul NC, Moodley A, Ghibaudo G, et al. Carriage of methicillin-
resistant Staphylococcus pseudintermedius in small animal vet-
erinarians: indirect evidence of zoonotic transmission. Zoonoses
Public Health. 2011;58:533-539.
29. Morris DO, Boston RC, O’Shea K, et  al. The prevalence of car-
riage of meticillin-resistant staphylococci by veterinary derma-
tology practice staff and their respective pets. Vet Dermatol.
2010;21(4):400-407.
CHAPTER 36
Gram-negative Bacterial Infections
Jane E. Sykes
Overview of Gram-negative Bacteria
First Described: Gram-negative bacteria were first described in
Berlin by the Danish scientist Hans Christian Gram in 1884
when he used Gram stain to visualize ­Klebsiella ­pneumoniae
in the lungs of people who died of pneumonia.1
Causes: Examples include Escherichia coli, Salmonella, Entero-
bacter, Citrobacter, Klebsiella, Campylobacter, Pseudomonas,
and Acinetobacter.
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact or contact with conta­m­
inated fomites; the bacteria often invade opportunistically
Major Clinical Signs: Affected sites include UTIs, wound
infections, pneumonia, pyothorax, peritonitis, pyometra,
ocular surface infections, otitis externa, pyoderma, bacte-
remia, and endocarditis, but virtually any body system can
be affected.
Differential Diagnoses: Infections with gram-positive bac-
teria such as Staphylococcus spp. and Streptococcus spp.;
to a lesser extent infections with nonbacterial pathogens
such as fungi.
Human Health Significance: Gram-negative bacteria shed
by dogs and cats have the potential to colonize humans,
especially those with impaired host defenses. They may
also be responsible for infections of dog or cat bites.
Etiology, Epidemiology, and Clinical Features
Gram-negative bacteria are widespread causes of opportunistic
infections in dogs and cats and primarily cause disease when
host defenses are impaired. They are important causes of noso-
comial disease. In contrast to gram-positive bacteria, gram-­
negative bacteria have a complex outer membrane that contains
lipopolysaccharide (LPS), as well as structures known as porins
that regulate transport of molecules in and out of the cell, which
includes antimicrobial drugs. Between this outer membrane and
the inner (cytoplasmic) membrane is a periplasmic space, which
contains the structural polymer peptidoglycan (or murein)
(­Figure 36-1). The peptidoglycan layer of gram-negative bacte-
ria is much thinner than that of gram-positive bacteria, approx-
imately one layer, or 7 to 8 nm thick (compared with many
layers, or 20 to 80 nm thick for gram-positive bacteria). The
inner cytoplasmic membrane contains many different proteins
that are lipoproteins anchored into the outer lipid bilayer leaflet,
transmembrane proteins, or peripheral membrane proteins that
lie adjacent to the bilayer leaflets.
LPS is a large molecule that varies in composition from one
bacterial species and strain to another. It contributes to the struc-
tural integrity of gram-negative bacteria and is a potent virulence
factor. It consists of the lipid A backbone, a core ­oligosaccharide,
and the O antigen side chain. Lipid A is a phosphorylated disac-
charide to which long, hydrophobic fatty acid chains are attached,
which anchor the LPS into the outer membrane (­Figures 36-1
and 36-2). The lipid A component is also known as endotoxin
because it is the biologically active portion of the molecule, in that
it stimulates a potent host inflammatory response (see Chapter
86). The core oligosaccharide connects lipid A to the O ­antigen
side chain. Its composition differs between bacterial species. The
O antigen is a repeating polysaccharide that projects from the
surface of the bacterial cell and varies in length from 1 to 60
repeats. It is the antigenic portion of the molecule and is respon-
sible for serogroup classification of gram-negative bacteria.
Gram-negative cocci, such as Moraxella or Neisseria, are not
widely recognized as significant pathogens of dogs and cats. How-
ever, they are commensals of the oral cavity of dogs and cats, can
cause bite wound infections in humans,2 and ­Neisseria canis was
isolated in pure culture from a deep mandibular abscess from one
dog.3 Gram-negative rods are classified into Enterobacteriaceae
and non-Enterobacteriaceae species. Enterobacteriaceae that
cause disease in dogs and cats include E ­ scherichia coli, ­Proteus,
­Salmonella, ­Enterobacter, ­Citrobacter, Serratia, and Klebsiella.
Non-Enterobacteriaceae species include the Pasteurellaceae
(­Pasteurella multocida), as well as P ­ seudomonas aeruginosa and
Acinetobacter. Other gram-negative bacteria are coccobacilli
or spiral-shaped organisms and include B ­ artonella, ­Bordetella,
­Campylobacter, ­Francisella, Helicobacter, and Brucella.
Like gram-positive cocci, some gram-negative bacteria have
tremendous ability to form biofilms, or bacterial aggregates
that are embedded in a matrix of exopolysaccharide (bacterial
slime), which form on living or nonliving surfaces. These pro-
tect the bacteria, and bacteria in biofilms can resist the effect of
antimicrobial drugs and disinfectants.
Enterobacteriaceae
Many of the Enterobacteriaceae contribute to the normal gastro-
intestinal flora of animals. Organisms that belong to the Entero-
bacteriaceae possess a capsule (K antigen) and may be motile via
flagella (H antigen). Some highly encapsulated species, such as
Klebsiella and Enterobacter, have a mucoid appearance when
they grow in culture. The flagella project from the bacterial
cell and contain the filament-forming protein flagellin, which
(along with LPS) can stimulate the host inflammatory response.
The Enterobacteriaceae also possess fimbriae or pili, which are
thinner than flagella and play a role in adhesion and sometimes
bacterial aggregation. Finally, they possess a variety of complex
355
356 SECTION 2  Bacterial Diseases
A B
FIGURE 36-1  Structure of the envelope of gram-positive (A) and gram-negative (B) bacteria. The thickness of the peptidoglycan layer is greater in gram-positive bacteria than in
gram-negative bacteria.
secretion systems (type I, II, III secretion systems). These facili-
tate export of toxins such as hemolysins, and molecules that
are inserted into, and form pores in, host cell membranes. The
bacteria then use these pores to inject toxins directly into the
host cell. The Enterobacteriaceae also have a variety of iron
scavenging systems, which are important virulence factors, and
transmissible plasmids that contain genes for virulence factors
and antimicrobial resistance factors.
Escherichia coli
Escherichia coli is the most common cause of urinary tract
infections (UTIs) in dogs and cats and can also be isolated from
dogs and cats with pyometra, prostatitis, peritonitis, cholangi-
tis, cholecystitis, bacteremia and endocarditis, bronchopneumo-
nia (such as that secondary to aspiration), and pyothorax. There
is one report of necrotizing fasciitis in a dog associated with E.
coli infection.4
Antibiotic resistance in E. coli isolates from dogs and cats
is an increasing problem; 51% of 376 isolates collected from
sick dogs and cats in the United States in 2005 showed resis-
tance to at least one drug, and 29% of the 376 isolates were
multiple drug resistant (MDR; defined as resistant to three or
more classes of antimicrobial drugs).5 The prevalence of resis-
tance in 395 isolates from healthy dogs and cats from Canada
was lower, 19%.6 Extended-spectrum β-lactamase enzyme pro-
duction has also been identified in E. coli isolates from dogs
and cats in the United States,7 and a few isolates were found
to express New Dehli metallo-β-lactamase (NDM-1), which
hydrolyses carbapenems.8 Nosocomial infections with MDR E.
coli have also been described in dogs and cats.
In humans, strains of E. coli that cause UTIs differ from those
that cause diarrhea, and there is some evidence that this may also
be true for strains that cause UTIs versus pyometra in dogs and
cats.9-11 Strains of E. coli that cause UTIs belong to the pathotype
extraintestinal pathogenic E. coli (ExPEC). These strains possess
a number of virulence factors, such as siderophores, type 1 and
P fimbriae, hemolysin, and cytotoxic necrotizing factor. Specific
virulence factors, as well as biofilm-forming capacity, have been
associated with persistent or relapsing UTIs,12,13 and uropatho-
genic E. coli can invade and persist within bladder epithelial
cells. In one study, canine UTIs associated with E. coli were more
likely to be recurrent than those associated with other uropatho-
gens.14 There has been concern that dogs may act as a source
of E. coli strains that are pathogenic for humans.11,15 In other
extraintestinal E. coli infections, E. coli more closely resemble
commensal strains, and host factors may be more important
than bacterial virulence factors. The reader is referred to Chapter
46 for more information on intestinal E. coli infections.
Proteus
Proteus mirabilis is a common cause of upper and lower UTIs
in dogs as well as otitis externa and uncommonly pyoderma.
It produces a potent urease and so may contribute to devel-
opment of struvite urolithiasis. Uroliths harbor bacteria that
can emerge as the uroliths dissolve. Proteus has a character-
istic swarming motility and may obscure the growth of other
co-infecting microorganisms when grown in the laboratory on
agar. ­Proteus penneri, an uncommon cause of nosocomial infec-
tions in humans, has rarely been isolated from wound infections
in dogs seen at the author’s hospital.
Klebsiella
The most common species of Klebsiella that infects dogs and
cats is K. pneumoniae. K. oxytoca is a nosocomial pathogen in
humans and has been reported in association with intravenous
catheter colonization in puppies with parvoviral enteritis.16
K. pneumoniae primarily causes upper and lower UTIs in
dogs and cats, but can also cause pneumonia, sometimes with
abscessation (Figures 36-3 and 36-4). It has also been isolated
from dogs with hepatic abscesses.17 Nosocomial infections may
be associated with bacteremia, endocarditis, and postoperative
skin and soft tissue infections. The capsule of K. pneumoniae is
 CHAPTER 36  Gram-negative Bacterial Infections
FIGURE 36-2  Structure of bacterial lipopolysaccharide.
a major virulence factor; it inhibits phagocytosis and is respon-
sible for the mucoid appearance of the organism in culture
(­Figure 36-5, A).
All strains of K. pneumoniae are resistant to ampicillin,
because they possess a chromosomal gene that encodes a penicillin
β-lactamase. Some isolates, such as nosocomial isolates or those
from persistent UTIs, possess plasmids that contain resistance
genes for multiple antimicrobial drugs. Of greatest concern are
Klebsiella isolates that express extended-spectrum β-lactamase
(ESBL) enzymes, which hydrolyze third-generation cephalospo-
rins such as cefotaxime or ceftriaxone. The genes that encode
ESBLs are often located on plasmids that also contain genes for
aminoglycoside resistance. Although MDR K. pneumoniae are
not uncommonly isolated from dogs and cats, ESBL-producing
K. pneumoniae isolates are rarely encountered. Treatment options
for MDR K. pneumoniae may be limited to aminoglycosides, car-
bapenems, and sometimes trimethoprim-sulfamethoxazole.
Serratia marcescens
Serratia marcescens is an important cause of nosocomial infections
in both human and veterinary medicine. In human patients it is
357
FIGURE 36-3  Lungs of an 11-year-old intact male German shepherd dog with
severe, acute, suppurative, necrotizing and hemorrhagic bronchopneumonia due to Kleb­
siella pneumoniae infection. The lung lobes are severely congested. Five days before death,
multiple laminectomies had been performed for intervertebral disc disease, and 2 days
later the dog developed aspiration pneumonia that necessitated mechanical ventilation.
Euthanasia was elected when there was further deterioration in the dog’s condition, and
bloody exudate was present in the endotracheal tube just before euthanasia.
FIGURE 36-4  Histopathology of the lung from a 4-month-old intact male New-
foundland dog with severe, multifocal, necrotizing lung abscesses. Intralesional rod-shaped
­bacteria were also present. Klebsiella pneumoniae was isolated from the lesions. H&E stain,
40× magnification.
often linked to intravenous drug use. The organism has a tremen-
dous ability to survive in the environment and may contaminate
and remain viable in disinfectant solutions.18 It sometimes grows as
a contaminant, such as in blood cultures. Some, but not all, strains
of S. marcescens produce a red pigment when grown in culture.
In dogs, S. marcescens infection has been associated with
UTIs, bacteremia and aortic valve endocarditis,19 catheter-
related infections in canine parvoviral enteritis,14 and necro-
tizing fasciitis.20 Contamination of blood products was linked
to an outbreak of bacteremia in cats.21 Serratia is resistant to
ampicillin and first-generation cephalosporins as a result of a
chromosomal β-lactamase. It may also acquire plasmid-mediated
resistance to other antimicrobial drugs.
Enterobacter
Enterobacter cloacae and, to a lesser extent, Enterobacter aero-
genes represent the majority of Enterobacter species isolated
358 SECTION 2  Bacterial Diseases

A B C
FIGURE 36-5  Gram-negative aerobic bacteria growing on blood agar. A, Mucoid colonies of Klebsiella pneumoniae. B, Pseudomonas aeruginosa. Transillumination using a light box
revealed the presence of hemolysis and production of a green pigment (pyoverdin). C, The plate in (B) had a fruity odor and a “mother-of-pearl” appearance when not transilluminated.

from dogs and cats in the author’s hospital. Enterobacter spp.

are mainly isolated from dogs and cats with UTIs but occasion-
ally cause wound or catheter-related infections. They generally
are intrinsically resistant to ampicillin and first-generation ceph-
alosporins and may also be resistant to multiple other antimi-
crobial drug classes.

Citrobacter
Citrobacter species are named for their ability to use citrate as a
sole carbon source. Citrobacter freundii is most commonly iso-
lated from dogs and cats with UTIs but can also cause catheter-
related infections, bacteremia, and endocarditis.22,23 ­Citrobacter
koseri was isolated from puppies with myocarditis.24 Like F
­Enterobacter and Serratia, Citrobacter are usually resistant to A
ampicillin and first-generation cephalosporins and may acquire
a number of plasmid-mediated resistance genes, including those
that encode ESBLs and resistance to fluoroquinolones.

Non-Enterobacteriaceae
Pasteurella
Bacteria that belong to the family Pasteurellaceae are small, fas-
tidious, nonmotile bacilli or coccobacilli that are commensals of
the oral cavities and upper respiratory tracts of dogs and espe-
cially cats. The most common species of Pasteurella associated P
with disease in dogs and especially cats is Pasteurella multocida.
Other species include Pasteurella canis, Pasteurella dagmatis,
and Pasteurella stomatis. Pasteurella spp. have been associated
with a wide variety of disease manifestations that include pyo- B
thorax, upper respiratory tract infections (usually secondary to
viral infections or chronic rhinosinusitis), bronchopneumonia, FIGURE 36-6  Electron micrograph of Pseudomonas aeruginosa showing ultrastructural
UTIs, ocular surface infections, wound infections, cutaneous features. A, Single cell with polar flagellum (F) with part of the flagellum running under the
abscesses, otitis externa, bacteremia, and, rarely, infective endo- cell body. B, Two cells showing thin, hair-like pili (P). (A, Courtesy of Dr. Steve Lory. In Mandell
GL, Bennett JE, Dolin R, eds. Mandell, Douglas and Bennett’s principles and practice of infec-
carditis. They may be found in mixed infections with other bac-
tious diseases, 7 ed. Philadelphia; Churchill Livingstone Elsevier; 2010:2835-2860; B, Courtesy
teria, such as Streptococcus canis, other gram-negative bacteria, of Dr. Martin Lee and Dr. Milan Bajmoczi, Harvard Medical School. In: Longo D, Fauci A, Kasper
or anaerobes. In humans, they are an important cause of animal D, et al. Harrison’s principles of internal medicine, 18 ed. Vol 1, New York; McGraw-Hill; 2012.)
bite wound infections.2 Pasteurella spp. are usually broadly sus-
ceptible to antimicrobial drugs, and the treatment of choice is a
penicillin such as penicillin G, ampicillin, or amoxicillin.
(blue), pyorubin (red), pyomelanin (black), and pyoverdin (yel-
Pseudomonas aeruginosa low-green to yellow-brown and fluorescent), and for its charac-
Pseudomonas aeruginosa is the main pathogenic species in the teristic “corn taco” or “grape”-like odor (see Figure 36-5, B and
family Pseudomonaceae. It is known for its ability to produce a C). P. aeruginosa has one polar flagellum and is covered in thin,
variety of pigments when grown in culture, such as pyocyanin hairlike pili (Figure 36-6). It can also produce an extracellular
 CHAPTER 36  Gram-negative Bacterial Infections
polysaccharide known as alginate, which results in a mucoid
colony appearance.
Pseudomonas aeruginosa is widespread in the environment
and can grow in a huge variety of different conditions, reflect-
ing its importance as a nosocomial pathogen. It is commonly
found on plants, water, and soil. It can survive in disinfectants
and grow in diesel and jet fuel. It is occasionally isolated from
the skin, mucous membranes, and feces of healthy animals and
humans. Almost any site of the body that is subject to injury
can become colonized and subsequently infected. In humans,
community-acquired infections can occur as a result of expo-
sure to contaminated hot tubs, swimming pools, and contact
lens solutions. In the hospital environment, P. aeruginosa
often colonizes moist environments such as sinks, endotracheal
tubes, and ventilator equipment. It also colonizes damp, mac-
erated tissues such as the ear canals, damaged cornea, trau-
matized skin (such as wounds and burns), and the interdigital
space. Because the organism can be resistant to a large number
of different antimicrobial drugs, repeated treatment of second-
ary bacterial infections in sites where impaired host defenses
exist with a variety of antimicrobials can select for MDR P.
aeruginosa infection at these sites. Examples of such conditions
in dogs and cats include recurrent aspiration pneumonia and/
or bronchiectasis, cystitis, chronic feline rhinosinusitis, and oti-
tis externa/media.
Pseudomonas aeruginosa possesses virtually every type
of bacterial virulence factor, including exotoxins, a type III
secretion system, lipopolysaccharide, pili, flagella, several
proteases, phospholipases, iron-scavenging mechanisms such
as pyoverdin production, and the ability to form extensive
biofilms. Alginate protects the organism from phagocytosis,
and the pigment pyocyanin produces reactive oxygen species,
which in turn damage host cells. Multidrug resistance results
from the production of specific β-lactamase enzymes (which
can include ESBLs), antibiotic efflux pumps, enzymes that
modify aminoglycosides or alter antibiotic binding sites (such
as DNA gyrase for fluoroquinolones), and decreased bacterial
permeability.
Infections with broadly susceptible P. aeruginosa isolates
could be treated with an antipseudomonal β-lactam (e.g., ticar-
cillin, a carbapenem, cefepime, ceftazidime) alone or in com-
bination with an aminoglycoside or a fluoroquinolone. The
combination of an aminoglycoside and an antipseudomonal
penicillin has synergistic activity against some P. aeruginosa
infections. Currently, most MDR P. aeruginosa isolates from
dogs and cats remain susceptible to carbapenems (such as
meropenem) and aminoglycosides (especially amikacin), and
so the usual treatment of choice is 1) a combination of these
two drugs, 2) removal of any foreign or necrotic material and,
3) if possible, resolution or management of any other underlying
cause for impaired host defenses. Because parenterally admin-
istered aminoglycosides do not effectively penetrate airway
mucus, nebulized tobramycin has been used to treat resistant
P. aeruginosa infections of the airways in human patients, but
these may not adequately penetrate consolidated lung tissue.25
Effective treatment of MDR P. aeruginosa infections when
they occur in locations such as the feline nasal cavity may be
extremely difficult, if not impossible, because of irreversible tur-
binate loss, the requirement for injectable antimicrobial drugs,
poor penetration of aminoglycosides to the site of infection, and
an inadequate ability to effectively treat all surfaces of the nasal
cavity with topical aminoglycosides.
359
Investigation of suspected nosocomial outbreaks of
P. aeruginosa infection generally requires typing of isolates
from affected animals and the environment with molecular
methods such as pulsed-field gel electrophoresis or multi-
locus sequence typing. Use of antibiograms alone does not
adequately discriminate between strains.
Acinetobacter
Like P. aeruginosa, Acinetobacter is an important nosoco-
mial opportunistic pathogen. Acinetobacter is present in soil
and water and can be found in frozen foods, pasteurized milk,
and an enormous array of inert objects in the hospital environ-
ment, where it may persist for months. It colonizes the skin and
mucous membranes of healthy humans as well as those of dogs
and cats.26,27 The main species that cause disease in animals are
Acinetobacter baumannii and, to a lesser extent, ­Acinetobacter
lwoffii and Acinetobacter calcoaceticus. Multi­drug resistant
isolates of A. baumannii have been responsible for outbreaks
of nosocomial infections in veterinary hospitals28,29 and, like
MDR P. aeruginosa, may be resistant to a huge variety of anti-
microbial drug classes. Carbapenem-resistant A. ­baumannii
have been isolated from humans as well as animals.29,30 Clini-
cal manifestations of Acinetobacter infections in dogs and
cats include nosocomial UTIs (e.g., after invasive urinary pro-
cedures such as catheterization), surgical wound infections,
pneumonia, catheter-related bacteremia, and, rarely, endocar-
ditis. Necrotizing fasciitis associated with A. baumannii infec-
tion was described in a cat.31 When Acinetobacter is grown
from a specimen, care should be taken to differentiate infection
from contamination. Most veterinary isolates are susceptible
to aminoglycosides and carbapenems; some are susceptible to
­trimethoprim-sulfamethoxazole and fluoroquinolones.28
Diagnosis
Laboratory Abnormalities
Complete Blood Count, Biochemistry, and Urinalysis Findings
Dogs and cats with superficial gram-negative infections of sites
that drain to the exterior, such as wounds, the external ear canal,
the nasal cavity, and the lower urinary tract, often have no clini-
cally significant laboratory abnormalities. Deep tissue or sys-
temic infections with gram-negative bacteria may be associated
with marked neutrophilia and a left shift, toxic neutrophils, or
a degenerative left shift, sometimes with circulating metamyelo-
cytes. Thrombocytopenia may be present in dogs and cats that
develop disseminated intravascular coagulation (DIC). Anemia
of inflammatory disease, lymphopenia, and monocytosis may
be present. The urinalysis of dogs and cats with gram-­negative
bacterial UTIs may reveal pyuria, proteinuria, hematuria, and
sometimes bacterial rods on sediment examination.
Coagulation Profile
Systemic gram-negative bacterial infections may be associated
with evidence of DIC due to activation of the coagulation cas-
cade by endotoxin.
Cytologic Abnormalities
Aspirates of affected tissues or pus, or transtracheal wash or
bronchoalveolar lavage fluid specimens from dogs and cats with
gram-negative bacterial infections may contain large numbers
of degenerate and nondegenerate neutrophils, sometimes with
bacterial rods.
360 SECTION 2  Bacterial Diseases

TABLE 36-1
Examples of Systemic Antimicrobial Drugs That Could Be Selected for Treatment of Gram-Negative Bacterial Infections
in Dogs and Cats (See Also Chapter 8)
Infecting Species Drug Dose (mg/kg) Interval (hours) Route
Pasteurella Amoxicillin 20 8 PO
Ampicillin 20 6-8 IV, IM, SC
Penicillin G 20,000-40,000 U/kg 6-8 IV, IM
Susceptible Trimethoprim-­sulfamethoxazole 30 12 PO, IV
­Enterobacteriaceae Amoxicillin–clavulanate 20 8 PO
­potassium
Ciprofloxacin 20-30 24 PO
10 24 IV
MDR ­Enterobacteriaceae, Amikacin* 15-30 (dogs), 10-14 (cats) 24 IV, IM, SC
Pseudomonas, or Gentamicin* 9-14 (dogs), 5-8 (cats) 24 IV, IM, SC
­Acinetobacter Ticarcillin disodium, ticarcillin- 33-50 4-6 IV
clavulanate
Meropenem 12 8 SC
25 8 IV
Ceftazidime 30 6 IV

*Use aminoglycosides in combination with an antipseudomonal β-lactam such as ticarcillin, meropenem, or a third-generation cephalosporin.

Microbiologic Tests amoxicillin, or clavulanic acid–amoxicillin are reasonable initial

Isolation and Identification
Gram-negative bacteria are readily isolated from aspirates, tis-
sue biopsies, body fluid, blood, and lavage specimens. MacCon­
key and blood agar are usually used to isolate gram-negative
bacteria, which usually grow within 24 to 48 hours and are sub-
sequently differentiated on the basis of colony morphology and
biochemical reactions. Pasteurella spp. are more fastidious, and
species that infect dogs and cats do not grow on MacConkey
agar. Evaluation of antimicrobial susceptibility is important for
all Enterobacteriaceae, P. aeruginosa, and Acinetobacter spp.,
given their propensity to develop resistance to many antimicro-
bial drug classes.
Treatment and Prognosis
The approach to treatment of gram-negative infections depends
on the site of infection and the severity of disease (Table 36-1); the
reader is referred to Chapters 84 to 90 for treatment recommenda-
tions for different organ systems. Identification and management
of the underlying cause are also important to minimize selection
for resistant organisms. Any foreign material (such as catheters,
implants) must be removed, and necrotic tissue debrided. When
systemic antimicrobial drug treatment is required, it should pref-
erably be based on the results of susceptibility testing, although
Pasteurella spp. are generally susceptible to penicillin or amino­
penicillins such as amoxicillin. Because gram-negative bacte-
ria have higher minimum inhibitory concentrations (MICs) to
penicillins than do gram-positive bacteria, the high end of the
dose range and more frequent administration may be necessary.
Some Enterobacteriaceae are intrinsically resistant to ampicillin
and first-generation cephalosporins, and initial treatment of seri-
ous infections while awaiting the results of susceptibility testing
should include an aminoglycoside, a third-generation cephalo-
sporin, or a fluoroquinolone. Trimethoprim-sulfamethoxazole,
choices for treatment of UTIs associated with bacterial rods.32
Treatment should also be based on knowledge of local resistance
patterns among gram-negative bacteria, which may vary from
region to region and practice to practice.
Prevention
Prevention of gram-negative infections involves resolution of
impairment in host defenses and strict hospital infection control
practices (see Chapter 11).
Public Health Aspects
Strains of E. coli and other MDR gram-negative bacteria
harbored by dogs resemble pathogenic strains isolated from
humans.15,30 As a result, concern has been raised that compan-
ion animals may be a source of organisms for human infection,
especially those that reside with humans with compromised
host defenses.
Pasteurella multocida is the most commonly isolated organ-
ism from human dog and cat bite wounds.2 Pasteurella spp. can
also colonize the respiratory tracts of humans with underlying
respiratory disease such as bronchiectasis or chronic sinusitis,
and they occasionally have been reported in association with
peritonitis, bacteremia, and endocarditis. Most of these humans
have had contact with domestic animals in the household, and
isolates from affected humans have been shown by molecu-
lar methods to be identical to those colonizing animals in the
household.33
Thus, immunocompromised owners or those with impaired
anatomic barriers should be instructed to wash their hands after
handling their pet and perform regular household cleaning and
disinfection. Companion animals should not be allowed to lick
open wounds or the owner’s face.
 CHAPTER 36  Gram-negative Bacterial Infections
CASE EXAMPLE
Signalment: “Lottie”, a middle-aged, female spayed domestic
shorthair from northern California
History: Lottie was evaluated for chronic nasal discharge. Since
her rescue as a stray 2.5 years earlier, intermittent sneezing
and bilateral mucopurulent nasal discharge had been noted,
and the discharge sometimes contained blood. Ten months
before the evaluation, Lottie had been anesthetized for a nasal
flush at a local veterinary clinic, but developed respiratory
distress on recovery and was hospitalized with oxygen
supplementation for 24 hours. She was ataxic, leaned to one
side, and did not eat well for days, but over the subsequent
months the ataxia and leaning gradually resolved. For the two
weeks before the evaluation Lottie had stertorous respiration
and intermittent episodes of open-mouth breathing. For the
past 9 months, Lottie had been treated with marbofloxacin
and prednisolone. Before that, treatments for the nasal
discharge included clavulanic acid–amoxicillin, doxycycline,
or azithromycin, as well as l-lysine supplementation. Lottie
lived with six other cats, all of which had access to the
outdoors, and she was fed commercial canned and dry cat
food. The FeLV and FIV status of these cats was unknown.
Current medications: marbofloxacin (5 mg/kg PO q24h) and
prednisolone (1 mg/kg PO q24h)
Physical Examination:
Body Weight: 4 kg
General: Bright, alert and responsive, hydrated. T = 100.3°F
(37.9°C), P = 220 beats/min, purring, body condition score 7/9.
Eyes, Ears, Nose, and Throat: Mucopurulent nasal discharge
was present that was most profuse on the left side. Nasal
airflow was absent on the left side. Mild dental calculus was
present, but there were no oral masses or ulceration.
Respiratory: An inspiratory stertor was present, and referred
upper airway noises were detected on auscultation of the
thorax.
Cardiovascular, Gastrointestinal and Genitourinary, and
Lymph Nodes: No clinically significant abnormalities were
detected. It was difficult to palpate abdominal structures
due to the presence of abdominal fat.
Neurologic Examination: There was a mild, intermittent, left-
sided head tilt. No other abnormalities were detected on full
neurologic examination.
Laboratory Findings:
CBC:
HCT 39.1% (30%-50%)
MCV 44.7 fL (42-53 fL)
MCHC 29.9 g/dL (30-33.5 g/dL)
Reticulocytes 111,400 cells/µL (7000-60,000 cells/µL)
WBC 21,210 cells/µL (4500-14,000 cells/µL)
Neutrophils 16,713 cells/µL (2000-9000 cells/µL)
Lymphocytes 4115 cells/µL (1000-7000 cells/µL)
Monocytes 297 cells/µL (50-600 cells/µL)
Eosinophils 64 cells/µL (150-1100 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL)
Platelets 316,000/µL (180,000-500,000 platelets/µL).
Serum Chemistry Profile:
Sodium 151 mmol/L (151-158 mmol/L)
Potassium 3.7 mmol/L (3.6-4.9 mmol/L)
Chloride 113 mmol/L (117-126 mmol/L)
Bicarbonate 19 mmol/L (15-21 mmol/L)
361
Phosphorus 5.8 mg/dL (3.2-6.3 mg/dL)
Calcium 10.2 mg/dL (9.0-10.9 mg/dl)
BUN 49 mg/dL (18-33 mg/dL)
Creatinine 1.4 mg/dL (1.1-2.2 mg/dL)
Glucose 128 mg/dL (63-118 mg/dL)
Total protein 8.7 g/dL (6.6-8.4 g/dL)
Albumin 3.6 g/dL (2.2-4.6 g/dL)
Globulin 5.1 g/dL (2.8-5.4 g/dL)
ALT 33 U/L (27-101 U/L)
AST 21 U/L (17-58 U/L)
ALP 36 U/L (14-71 U/L)
Gamma GT 0 U/L (0-4 U/L)
Cholesterol 192 mg/dL (89-258 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL).
Urinalysis: USG 1.044, pH 6, 75 mg/dL protein, no glucose,
bilirubin or ketones, 250 erythrocytes/µL hemoprotein,
>100 RBC/HPF (traumatic cystocentesis), 0-1 WBC/HPF.
Imaging Findings:
Thoracic Radiographs: Cardiovascular structures were within
normal limits. There was a small amount of air in the cervical
esophagus (likely due to aerophagia) and a mild diffuse
bronchointerstitial pattern that was attributed to an obese
body condition.
Skull Computed Tomography Scan: Contiguous transverse
0.6-mm collimated images of the skull were available for
review without contrast administration. There was moderate
turbinate destruction of both right and left nasal passages
with lobular soft tissue attenuating material present along
their length (­Figure 36-7, A). There was stenosis of both
frontal sinuses to the point where the left frontal sinus was
completely occluded. The right frontal sinus was almost
completely filled with soft tissue attenuating material.
The ear canals were within normal limits bilaterally. The
mandibular lymph nodes were prominent but symmetrical.
Rhinoscopy: Severe turbinate destruction with diffuse
hyperemia and large accumulations of inspissated green
mucus was present throughout the nasal passages (­Figure
36-7, B). Several biopsies of the right and left nasal cavities
were obtained for histopathology.
Microbiologic Testing: Aerobic bacterial culture and
Mycoplasma culture (nasal brush specimen): a direct smear
showed moderate numbers of neutrophils, but no organisms
were seen. Moderate numbers of Pseudomonas aeruginosa
were isolated. The P. aeruginosa was resistant to amoxicillin–
clavulanic acid, ampicillin, cefazolin, cefovecin, cefoxitin,
cefpodoxime, ceftiofur, chloramphenicol, doxycycline,
enrofloxacin, marbofloxacin, ticarcillin, ticarcillin–clavulanic
acid, and trimethoprim-sulfamethoxazole. It was susceptible
only to amikacin (≤4 µg/mL), gentamicin (2 µg/mL), and
imipenem (≤1 µg/mL).
Histopathology of Nasal Biopsy Specimens: The
submucosa was markedly expanded and multifocally
effaced by sheets of lymphocytes and plasma cells,
with neutrophils and fewer numbers of mast cells
surrounding irregular spicules of woven bone extending
from primary turbinates. Less affected sections contain
similar, predominately lymphoplasmacytic infiltrates
that separated submucosal glands and elevated the
superficial epithelium. Pathologic diagnosis: severe, chronic,
and extensive lymphoplasmacytic and neutrophilic
rhinitis with turbinate remodeling.
Continued
362 SECTION 2  Bacterial Diseases
L R
A A
FIGURE 36-7  Nasal cavity of a middle-aged, female spayed domestic shorthair with chronic rhinosinusitis and secondary infection with multidrug-resistant Pseudomonas aeru­
ginosa. A, Computed tomographic scan showing destruction of the turbinates of the left and right nasal passages. B, Rhinoscopic findings included severe turbinate destruction,
hyperemia, and accumulations of thick green mucus.
Diagnosis: Feline chronic rhinosinusitis with suspected
opportunistic MDR P. aeruginosa infection.
Treatment and Outcome: The nasal cavity was lavaged. The
prednisolone was tapered over 1 week, then discontinued,
and 7 days later, treatment with piroxicam was commenced
(0.3 mg/kg PO q24h for 7 days, then q48h thereafter). Other
medications discussed with the owner were N-acetylcysteine
and intranasal gentamicin drops, but the prognosis given for
disease resolution was poor.
Comments: The underlying cause of feline chronic rhinosinusitis
is poorly defined, but infection of kittens with respiratory
viruses such as feline herpesvirus 1 (FHV-1) is suspected
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Hohenhaus AE, Drusin LM, Garvey MS. Serratia marcescens contami-
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Assoc. 1997;210:794-798.
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29. Francey T, Gaschen F, Nicolet J, et al. The role of Acinetobacter
baumannii as a nosocomial pathogen for dogs and cats in an inten-
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30. Endimiani A, Hujer KM, Hujer AM, et  al. Acinetobacter bau-
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CHAPTER 37
Anaerobic Bacterial Infections
Jane E. Sykes
Overview of Anaerobic Bacterial Infections
First Described: 1860s (Louis Pasteur)1
Causes: Bacteroides, Prevotella, Porphyromonas, Peptostrep-
tococcus, Fusobacterium, Clostridium, Propionibacterium,
other species
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact, and opportunistic
invasion of tissues by commensal bacteria
Major Clinical Signs: Abscess formation in a variety of tissues,
pyothorax, peritonitis, pyometra, periodontal disease,
osteomyelitis, bacteremia
Differential Diagnoses: Primarily bacterial infections caused
by gram-positive and gram-negative aerobes, but also
neoplasia (when discrete abscessation is present).
Human Health Significance: Anaerobes of the oral cavities of
dogs and cats are an important component of animal bite
wound infections in humans. Enteric clostridial infections
of dogs and cats may also have public health significance
(see Chapter 48).
Etiology and Epidemiology
An anaerobe is an organism that requires an environment that is
reduced in oxygen for growth and does not grow in air. Anaer-
obic bacteria represent a significant component of the normal
flora of the upper and lower gastrointestinal tracts of dogs and
cats as well as their genital tracts; in addition, anaerobes play an
important role in the regulation of the bacterial composition of
the normal flora. Aerobes and facultative organisms that reside
alongside anaerobes use oxygen present in these sites and reduce
the local oxidation-reduction potential, which permits survival
and growth of anaerobes. Sites that are protected from oxygen
such as the gingival crevices also favor anaerobes. The metabolic
activity of anaerobic bacteria prevents colonization of mucosal
surfaces by potentially more pathogenic microbes (this is known
as colonization resistance). Finally, anaerobes are important for
the maintenance of normal mucosal barrier function.
Anaerobic bacteria can cause disease when they gain access to
normally sterile sites. This occurs when mucosal barriers are dis-
rupted, or when anaerobes gain access to subcutaneous tissues as
a result of a contaminated penetrating wound. Anaerobic bacte-
rial infections are often mixed infections owing to the concurrent
presence of other anaerobes, as well as aerobes and facultative
bacteria such as Pasteurella spp., Escherichia coli, streptococci,
or staphylococci. Although there are hundreds of anaerobes
364
that comprise the normal flora, the anaerobic bacterial species
most often identified in disease are the gram-negative anaerobes
Fusobacterium spp., Bacteroides spp., Prevotella spp., and Por-
phyromonas spp., and the gram-positive anaerobes Clostridium
spp. and Peptostreptococcus spp. (Box 37-1).2 An accurate
understanding of the epidemiology of anaerobic bacterial infec-
tions in dogs and cats has been hampered by difficulties associ-
ated with culture of anaerobes. The use of molecular methods
such as PCR has begun to shed more light on this topic. The
reader is referred to Chapters 48 and 54 for information on
gastrointestinal Clostridium perfringens and Clostridium diffi-
cile infections, and disease caused by the toxins of Clostridium
­tetani and Clostridium botulinum, respectively.
Clinical Features
Signs and Their Pathogenesis
Anaerobes can cause disease in a variety of different organs.
Often, their presence is associated with abscess formation and
necrosis at the site of inoculation, which contains a mixed popu-
lation of anaerobic as well as facultative bacteria. The posses-
sion of virulence factors by anaerobes contributes to their ability
to cause disease after host barriers are disrupted. These include
capsular polysaccharides, extracellular proteases, endotoxin
(gram-negative anaerobes), and a variety of other toxins. Capsu-
lar polysaccharides play an important role in the stimulation of
BOX 37-1
Major Anaerobic Bacterial Pathogens Isolated
from Dogs and Cats
Gram-Positive Cocci
Peptostreptococcus spp. (especially Peptostreptococcus
anaerobius)
Gram-Positive Rods
Clostridium spp.
Propionibacterium spp. (especially Propionibacterium
acnes)
Gram-Negative Rods
Bacteroides spp. (e.g., Bacteroides fragilis)
Fusobacterium spp. (e.g., Fusobacterium necrophorum
and Fusobacterium nucleatum)
Prevotella spp. (e.g., Prevotella heparinolyticus)
Porphyromonas spp.

abscess formation by certain anaerobes such as Bacteroides, Por-
phyromonas, Fusobacterium, and Prevotella species. Just as aer-
obic bacteria potentiate the survival of anaerobes through local
consumption of oxygen, anaerobic bacteria produce metabolites
such as short-chain fatty acids, which inhibit phagocytosis and
promote survival of co-infecting facultative organisms.
Anaerobes of the oral cavity are important contributors
to periodontal disease, tooth root abscesses and mandibular
osteomyelitis, retrobulbar abscesses, and bite wound infections.
Anaerobes are frequently isolated from the lungs and pleural
cavities of dogs or cats with aspiration pneumonia and pyotho-
rax, respectively. Intestinal anaerobes may be isolated from the
abdominal cavities of animals with septic peritonitis secondary
to intestinal perforation. The female genital tract is also heav-
ily colonized with anaerobic bacteria, and pyometra may some-
times be associated with mixed aerobic and anaerobic infections
that include the anaerobic species Bacteroides, Prevotella, Fuso-
bacterium, and Peptostreptococcus species.
Plant awn migration in dogs and cats can lead to abscess for-
mation in a variety of tissues, including retroperitoneal tissues
and the brain. These abscesses usually contain a mixed popula-
tion of aerobes and anaerobes, especially Fusobacterium, Acti-
nomyces, and Bacteroides species. Brain abscessation can follow
direct extension of anaerobic bacterial infection from otitis or
tooth root infection. Alternatively, hematogenous spread may
occur (Figure 37-1).
Periodontal Disease
Periodontal disease is one of the most common disorders seen
by small animal veterinarians and may result in chronic pain,
decreased appetite, and loss of teeth. It begins with formation of
FIGURE 37-1  Abscess in the white matter of the right frontal lobe of a 2-year-old
female spayed Chihuahua that had a 1-month history of difficulty chewing food and
lethargy and a 3-week history of progressive obtundation, neck pain, and tetrapare-
sis. Signs initially improved following treatment with prednisone, but then recurred as
the prednisone dose was reduced. The abscess in the frontal lobe communicated with
the overlying meninges, which were expanded by a chronic and fibrosing suppurative
inflammatory infiltrate. Another abscess was present in the midbrain, which suggested
hematogenous spread of infection. Although no bacteria were seen within the lesions
with special stains, Actinomyces canis and Fusobacterium nucleatum were cultured
from the lesions. (Courtesy of the University of California, Davis Veterinary Anatomic  tact ulceration of the buccal mucosa are also present. (Courtesy of Dr. Frank Verstraete,
Pathology Service.)
CHAPTER 37  Anaerobic Bacterial Infections 365
dental plaque as a result of poor oral hygiene and other host fac-
tors. Dental plaque is a bacterial biofilm composed primarily of
gram-positive facultative anaerobes. Subsequently there is pro-
liferation of pathogenic bacteria, which include gram-negative
rods and especially anaerobes. These secrete toxins and incite
an inflammatory response. Anaerobes implicated in canine and
feline periodontal disease (such as Porphyromonas gulae) dif-
fer from those implicated in human periodontal disease (Por-
phyromonas gingivalis).3-6 Periodontal disease is manifested as
gingival erythema, edema, gingival bleeding, and halitosis, with
accumulation of calculus, gingival recession, alveolar bone loss,
and sometimes the presence of purulent exudate (Figure 37-2).
Retrobulbar Abscesses
Retrobulbar or orbital abscesses can result from tooth root
abscessation, penetrating foreign bodies such as grass awns, or
penetrating trauma to the orbit. Clinical signs include unilateral
exophthalmos, protrusion of the nictitans, conjunctival hyper-
emia, serous to mucopurulent ocular discharge, inappetence,
fever, and pain on opening the mouth (Figure 37-3). A fluctuant
swelling or draining tract posterior to the last ipsilateral molar
may be present on examination of the oral cavity. In one study,
Bacteroides spp. and Clostridium spp. were the most frequent
anaerobes isolated from 34 dogs with retrobulbar abscesses;
other frequently isolated bacteria were Staphylococcus spp.,
Pasteurella spp., and Escherichia coli. In cats, the most frequent
anaerobe was Bacteroides vulgatus, and the most common non-
anaerobe was Pasteurella spp.7
Skin and Soft Tissue Infections
Cat bite abscesses are one of the most common reasons that cats
are brought to small animal clinics. Clinical signs include fever,
lethargy, inappetence, pain, and firm or fluctuant subcutaneous
swellings that may rupture and drain purulent material. Oral
anaerobes and Pasteurella multocida are frequently isolated
from these abscesses.8 Anaerobic species involved include Pep-
tostreptococcus anaerobius, Porphyromonas spp., Clostridium
villosum, Fusobacterium spp., Bacteroides spp. (especially Bac-
teroides tectum), and Prevotella spp.9 Despite the widespread
occurrence of cat bite abscesses, studies that use molecular
methods to identify the types and relative prevalences of anaer-
obes in cat bite abscesses are lacking.
FIGURE 37-2  Periodontitis in a dog. There is significant gingival inflammation,
plaque and calculus accumulation, and purulent exudate. Gingival recession and con-
University of California, Davis Dentistry and Oral Surgery Service.)
366 SECTION 2  Bacterial Diseases
Other skin and soft tissue infections associated with a variety
of anaerobes include foreign body penetrating wounds and dog
bite wounds (see Chapter 57).
Pneumonia and Pyothorax
Anaerobes such as Fusobacterium spp., Peptostreptococcus
anaerobius, Prevotella spp., and Porphyromonas spp. are fre-
quently isolated from dogs and cats with aspiration pneumo-
nia, foreign body pneumonia (especially that due to plant awn
migration), and pyothorax.10 Dogs with plant awn migration
develop multifocal necrotizing pneumonia with abscess forma-
tion. In some cases, aspiration pneumonia is followed by lung
abscess formation (Figure 37-4). In human patients, a period
of 6 to 7 days is required for necrosis and abscess formation to
occur after aspiration, and so aspiration pneumonia with lung
abscess formation is generally a chronic disease process.
A
B
FIGURE 37-3  Retrobulbar abscess and endophthalmitis secondary to a migrating
grass awn foreign body in a 10-month-old intact male Labrador retriever. A, Note the
mucopurulent ocular discharge from the right eye, as well as buphthalmos (enlarge-
ment of the eye) and exophthalmos. The nictating membrane and conjunctiva are
hyperemic, and there is diffuse corneal edema. Hypopyon and iris bombe were also
present. Globe retropulsion was reduced and painful on the right side. B, Multiple grass
awns were present all over the dog’s haircoat, and an awn (shown) was removed from
the gingiva posterior to the last right maxillary molar tooth. The right eye was subse-
quently enucleated. No additional grass awns were found. Histopathology of the globe
showed severe chronic fibrinosuppurative panophthalmitis with focal, 0.3-cm scleral
rupture in the caudal sclera near the optic nerve, regional orbital granulation tissue,
and rupture of the lens. (Courtesy of the University of California, Davis Veterinary Oph-
thalmology Service.)
The reader is referred to Chapter 87 for more information on
bacterial pneumonia and pyothorax.
Peritonitis
Septic peritonitis that involves anaerobes may result from perfo-
ration of the intestinal tract, especially the cecum or colon. Bac-
teroides and Clostridium species are most often isolated from
these infections. Fusobacterium and Propionibacterium species
may also be isolated.11 Disease that results from bowel perfora-
tion often contains a population of organisms that reflect the
bowel flora at the site of perforation, including anaerobes, fac-
ultative gram-positive anaerobes (especially Enterococcus spp.),
Candida spp., and facultative gram-negative anaerobes such as
Escherichia coli. Rupture of the female genital tract secondary
to pyometra may also result in anaerobic bacterial peritonitis.
Bacteremia
Anaerobes are occasionally isolated from dogs and cats with
bloodstream infections, usually as polymicrobial infections with
aerobic bacteria. In a European study of 66 bacteremic cats,
12% of bacteria isolated from the bloodstream were obligate
anaerobes.12 In a study of 140 bacteremic dogs, anaerobes
comprised 9% of the isolates.13 Of 39 dogs and 10 cats with
critical illness and bacteremia from Colorado, anaerobes were
isolated from 31% of the dogs and 40% of the cats. The most
common anaerobe isolated from the 39 dogs was C. perfringens
(9 dogs), followed by Bacteroides species (2 dogs).14 The cats
were infected with Bacteroides or Fusobacterium species. The
possibility of mixed aerobic-anaerobic bacterial bloodstream
infection should be considered in dogs with systemic grass awn
migration. However, anaerobic bacteria appear to be a very rare
cause of endocarditis in dogs (see Chapter 86).15
FIGURE 37-4  Necropsy findings in an 11-year-old male neutered Labrador retriever
with severe aspiration pneumonia secondary to laryngeal tie-back surgery for laryngeal
paralysis, which occurred 2 months before death. Aspiration led to bronchopneumonia
with multifocal abscessation, pleural rupture, pyothorax, and pneumothorax. There are
multiple to coalescing black to dark gray, soft, irregular foci in the lungs that measured up
to 4.5 cm in diameter. On cut surface, dark gray to black material oozed from the paren-
chyma. Aerobic bacterial culture yielded large numbers of bacteria that included Strep-
tococcus canis, nonfermenter group 3 organisms, and Pasteurella canis. Anaerobic culture
yielded large numbers of Peptostreptococcus anaerobius. (Courtesy of the University of
California, Davis, Veterinary Anatomic Pathology Service.)

Diagnosis
Abnormalities that suggest an anaerobic bacterial infection
include a putrid odor, discolored purulent discharge, gas pro-
duction within tissues (especially with C. perfringens infections)
(Figure 37-5), a bite or puncture wound, penetrating foreign
bodies, abscessation, pyothorax, aspiration pneumonia, damage
to mucosal surfaces with which anaerobes are usually associ-
ated, and/or necrotic or gangrenous tissue (Box 37-2). How-
ever, absence of these features does not rule out infection with
anaerobes, and some of these abnormalities occur with pure
aerobic bacterial infections. For example, although anaerobes
such as Clostridium spp. may be involved in emphysematous
cystitis in humans and dogs, the most common cause of emphy-
sematous cystitis is E. coli.
Laboratory Abnormalities
Complete Blood Count, Biochemistry, and Urinalysis Findings
No specific CBC, biochemistry, or urinalysis findings suggest
anaerobic infection. Infections may be associated with leukocy-
tosis due to a neutrophilia with a left shift and toxic neutrophils.
Cytologic Analysis
Cytologic analysis of exudate in aerobic bacterial infections typ-
ically reveals neutrophilic inflammation accompanied by bacte-
ria of mixed morphology and gram staining characteristics. This
and/or the presence of filamentous rod-shaped bacteria within
these exudates should increase suspicion for an anaerobic infec-
tion (Figure 37-6).
Microbiologic Tests
Bacterial Isolation and Identification
Optimal techniques for isolation of anaerobic bacteria are
described in Chapter 3. The decision to request anaerobic bac-
terial culture should be considered carefully, because anaerobes
are difficult to culture, tend to have predictable susceptibility
to antimicrobial drugs, and can contaminate specimens, which
can lead to misleading results. If anaerobic culture is performed,
the best specimens to collect are tissues or body fluids that are
normally sterile. Fluids (e.g., 1 to 2 mL) or tissue specimens are
FIGURE 37-5  Splenic abscess in an 11-year-old male neutered Labrador mix that
also had duodenal lymphoma. Ultrasound image of a large splenic mass that contains
numerous hyperechoic foci and shadowing, consistent with the presence of gas. Necropsy
showed severe, focally extensive, neutrophilic, and necrotizing splenitis. The duodenum
was perforated at the site of the neoplasm. Escherichia coli, Bacteroides tectum, and a Pre-
votella heparinolyticus–like organism were cultured from the splenic abscess.
CHAPTER 37  Anaerobic Bacterial Infections 367
preferable to swab specimens. Specimens should immediately be
placed in an anaerobic transport medium and shipped to the
laboratory as soon as possible, because even brief exposure to
oxygen impairs isolation. Growth is often slow when compared
to other facultative organisms, and cultures should be incu-
bated for 7 days. Because most anaerobe infections also involve
aerobes, aerobic bacterial culture should always be requested
concurrently.
Extensive antimicrobial susceptibility testing is not rou-
tinely performed for anaerobes because it is laborious,
expensive, and can have a long turnaround time. Because
infections are polymicrobial (two or more anaerobe spe-
cies as well as facultative organisms present), susceptibility
testing for all pathogens present is often cost prohibitive to
BOX 37-2
Disease Processes That Suggest the Possibility of Anaerobic
Bacterial Infection
Abscess formation
Putrid odor
Necrotic tissue
Negative cultures despite the presence of bacteria
on cytologic examination
Exposure to plant foreign bodies such as grass awns
Interdigital or retrobulbar soft tissue involvement
Dental infections
Aspiration pneumonia
Pyothorax
Gastrointestinal perforation
Pyometra
Bite wound infections
FIGURE 37-6  Cytology of pleural fluid collected using thoracocentesis from an
8-year-old female spayed domestic shorthair with pyothorax. A mixed population of
bacteria that includes filamentous organisms suggestive of anaerobes (arrow) is present.
Aerobic and anaerobic bacterial culture yielded a mixed growth of Prevotella spp., Pepto-
streptococcus anaerobius, Fusobacterium spp., and an unidentified anaerobic gram-positive
rod. Wright’s stain, 1600× magnification.
368 SECTION 2  Bacterial Diseases

TABLE 37-1
Suggested Antimicrobial Drugs for Treatment of Anaerobic Bacterial Infections in Dogs or Cats*
Drug Dose (mg/kg) Interval (hours) Route
Amoxicillin–clavulanate potassium 12.5-25 12 PO
Ampicillin-sulbactam 10-20† 8 IV, IM
Metronidazole 15 12 IV
15 12 (dogs), 24 (cats) PO
Clindamycin hydrochloride, clindamycin phosphate 11 12 (dogs), 24 (cats) PO
10 12 IV‡, IM
Chloramphenicol, chloramphenicol sodium succinate 40-50 (dogs), 12.5-20 (cats) 6-8 (dogs), 12 (cats) PO, IV, IM§
Pradofloxacin 3 (dogs), 5-10 (cats) 24 PO∥

*The concurrent presence of aerobes should also be considered when selecting antimicrobial drugs.
†Dose according to ampicillin component.
‡For IV administration, dilute 1:10 in 0.9% saline and administer over 30-60 minutes.
§See Chapter 8 for warnings about chloramphenicol use.
∥Use of pradofloxacin in dogs at high doses has been associated with myelosuppression and is extra-label in the United States. Doses shown for cats

are for the oral suspension; a dose of 3 mg/kg is recommended for the tablet formulation.

pet owners. In addition, anaerobes that infect dogs and cats
generally have predictable drug susceptibilities. Neverthe-
less, because antimicrobial resistance has increased among
anaerobes that infect humans, the possibility of drug resis-
tance should be considered when infections do not respond to
treatment with drainage and appropriate antimicrobial drugs.
Some veterinary diagnostic laboratories perform rapid tests
for β-lactamase enzyme production by anaerobes (see Case
Example).
Treatment and Prognosis
Treatment of anaerobic bacterial infections involves surgical
removal of necrotic tissue, drainage of abscesses through surgi-
cal or imaging-guided approaches, restoration of blood supply,
and medical management with antimicrobial drugs (Table 37-1).
Antimicrobial drugs with activity against most anaerobes are
β-lactam/β-lactamase inhibitor drug combinations (such as cla-
vulanic acid–amoxicillin or ampicillin-sulbactam), clindamycin,
metronidazole, chloramphenicol, and carbapenems. Although
penicillin and first-generation cephalosporins may be effective,
some pathogenic anaerobes that infect dogs and cats (especially
Bacteroides but also a significant percentage of Prevotella, Por-
phyromonas, and Fusobacterium isolates) may be resistant to
these drugs through the production of β-lactamase enzymes.
Approximately 20% of Bacteroides and Clostridium isolates are
resistant to clindamycin, but other anaerobes are susceptible.2
Factors such as tissue penetration should be considered when
selecting antimicrobial drugs, and the concurrent presence of
aerobic bacteria must also be taken into account. Thus a combi-
nation of a β-lactam/β-lactamase inhibitor drug or clindamycin
and a drug with activity against gram-negative bacteria, such as
a fluoroquinolone or an aminoglycoside, is a reasonable choice
for initial treatment of serious infections while awaiting the
results of susceptibility testing for aerobic bacteria when mixed
aerobic-anaerobic infections are suspected. For infections that
do not require parenteral therapy, pradofloxacin also has potent
activity against a variety of aerobes, anaerobes and mycoplas-
mas (see Chapter 8).
With the exception of newer generation fluoroquinolones
such as pradofloxacin, anaerobes are resistant to fluoroquino-
lones. Anaerobes are also generally resistant to trimethoprim-
sulfamethoxazole and have unpredictable resistance to
tetracyclines. Aminoglycosides are remarkable for their com-
plete inactivity against anaerobes.
Prevention
Prevention of anaerobic bacterial infections involves manage-
ment or prevention of underlying conditions that predispose
to these infections. Vaccines for prevention of anaerobic bac-
terial infections are not available. An inactivated, whole-cell
Porphyromonas denticanis, Porphyromonas gulae, and Por-
phyromonas salivosa vaccine was available for several years for
prevention of periodontal disease in dogs but was withdrawn
from the market because of apparent lack of efficacy.
Public Health Aspects
Anaerobes that reside in the oral cavity of dogs and cats are
important causes of bite wound infections in humans, next
to Pasteurella species. The reader is referred to Chapter 57
for more information on the public health significance of bite
wound infections.

CASE EXAMPLE
Signalment: “Cocoa,” a 4-month-old intact female Labrador
retriever from Gonzales in northern California
History: Cocoa was brought to the University of California,
Davis, Veterinary Medical Teaching Hospital for
evaluation of seizures. Approximately 6 weeks before
referral, the dog was seen for left ocular exophthalmos.
She was treated with amoxicillin–clavulanic acid for
1 week, and the exophthalmos resolved, but subsequently
right ocular exophthalmos developed. This resolved after
another 1-week course of amoxicillin–clavulanic acid.
One month later, Cocoa appeared painful on opening her
mouth. She was treated with cephalexin and meloxicam
with no apparent improvement. Two days later she became
inappetent. When the owners offered her food by hand, she
ate, became ataxic, fell over, her thoracic limbs stiffened,
and foamy saliva was noted around her mouth. She was
taken to an emergency clinic, and she vomited in the car.
At the emergency clinic she had three generalized seizures
that were treated with diazepam. She was also treated with
enrofloxacin (5 mg/kg IV, once).
Cocoa’s diet normally consisted of commercial dog food, and
she had been vaccinated for distemper, hepatitis, and par-
voviral enteritis at 8 and 12 weeks of age. There had been
no coughing, sneezing, diarrhea, exposure to ticks or toxins,
trauma, or travel outside California.
Physical Examination:
Body Weight: 15.3 kg
General: Quiet but alert and responsive. Hydrated.
T = 102.8°F (39.3°C), HR = 110 beats/min, panting, mucous
membranes pink, CRT = 1 second.
Eyes, Ears, Nose, and Throat: No clinically significant
abnormalities were noted. There was no evidence of pain
on opening the mouth provided neck movement was
prevented during the examination.
Integument, Musculoskeletal, Cardiovascular, Gastro­
intestinal, Genitourinary Systems, and Peripheral
Lymph Nodes: No clinically significant findings.
Neurologic Examination: Mild obtundation was noted. The
dog held her head down and was reluctant to move her
neck. There was moderate to marked ataxia that involved all
four limbs, with the pelvic limbs most severely affected, and
the right pelvic and thoracic limbs more severely affected
than the left. Cranial nerve examination revealed only a
decreased corneal reflex on the right side. Decreased placing
reactions were noted in all four limbs, with the right limbs
more severely affected. There was severe pain on palpation
of the cervical spine and moderate pain on palpation of the
lumbosacral spine. Multifocal CNS lesions were suspected.
Laboratory Findings:
CBC:
HCT 34.4% (40%-55%)
MCV 64.9 fL (65-75 fL)
MCHC 34.9 g/dL (33-36 g/dL)
Reticulocytes 22,000 cells/µL (7000-65,000 cells/µL)
WBC 17,780 cells/µL (6000-13,000 cells/µL)
Neutrophils 12,979 cells/µL (3000-10,500 cells/µL)
Band neutrophils 533 cells/µL
CHAPTER 37  Anaerobic Bacterial Infections 369
Lymphocytes 3023 cells/µL (1000-4000 cells/µL)
Monocytes 1245 cells/µL (150-1200 cells/µL)
Platelets 392,000 platelets/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 143 mmol/L (145-154 mmol/L)
Potassium 4.6 mmol/L (3.6-5.3 mmol/L)
Chloride 102 mmol/L (108-118 mmol/L)
Bicarbonate 23 mmol/L (16-26 mmol/L)
Phosphorus 6.9 mg/dL (3.0-6.2 mg/dL)
Calcium 11.1 mg/dL (9.7-11.5 mg/dl)
BUN 7 mg/dL (5-21 mg/dL)
Creatinine 0.4 mg/dL (0.3-1.2 mg/dL)
Glucose 95 mg/dL (64-123 mg/dL)
Total protein 6.1 g/dL (5.4-7.6 g/dL)
Albumin 2.8 g/dL (3.0-4.4 g/dL)
Globulin 3.3 g/dL (1.8-3.9 g/dL)
ALT 35 U/L (19-67 U/L)
AST 52 U/L (19-42 U/L)
ALP 103 U/L (21-170 U/L)
Creatine kinase 556 U/L (51-399 U/L)
Gamma GT 4 U/L (0-6 U/L)
Cholesterol 262 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.013; pH 6.5, negative for protein, glucose,
ketones, bilirubin, hemoprotein, WBC, RBC, casts, and bacteria.
Imaging Findings:
Thoracic Radiographs: Cardiopulmonary structures were
within normal limits.
Abdominal Ultrasound: The gastric wall was mildly thickened
with bright, echogenic speckles within the muscularis
layer. The submucosa of the stomach was echogenic. These
changes were suggestive of gastritis. The remainder of the
abdominal ultrasound was unremarkable.
Spinal Radiographs: Open physes were present within the
vertebral column. No abnormalities were identified.
Brain MRI: In the precontrast brain study, a flattened, fusiform
extradural structure was identified in the dorsal part of
the foramen magnum that caused moderate dorsoventral
compression of the spinal cord (see arrow in Figure 37-7). This
structure was hypointense to brain but hyperintense to CSF
on the T1-weighted images and moderately hyperintense
on dual echo and fluid-attenuated inversion recovery
(FLAIR) images. The postcontrast brain study showed diffuse
meningeal enhancement that involved the spinal cord and
brain, with the most prominent enhancement noted along the
ventral brainstem and left cerebral hemisphere. This contrast
enhancement pattern was consistent with meningitis. A
discrete rim of enhancement surrounded the aforementioned
fusiform structure, which gave it a cystic appearance. The
structure had partial fluid characteristics, and, in light of the
meningeal enhancement, an abscess was suspected.
Analysis of CSF (Lumbar CSF): CSF protein: 726 mg/dL
(reference range, <25 mg/dL)
Total RBC: 5 cells/µL (reference range, 0 cells/µL)
Total nucleated cells: 7500 cells/µL (reference range,
<2 cells/µL)
Differential: 88% neutrophils, 12% large mononuclear cells
Microscopic evaluation: The specimen was highly cellular
and contained a large number of nondegenerate neutro-
phils. A few pyknotic forms were noted. Lower numbers of
Continued
370 SECTION 2  Bacterial Diseases
FIGURE 37-7  Postcontrast brain MRI, 4-month-old intact female Labrador
retriever with Bacteroides fragilis meningitis and an epidural abscess located in the
dorsal part of the foramen magnum. There is a flattened, fusiform structure in the
dorsal part of the foramen magnum associated with moderate dorsoventral compres-
sion of the spinal cord (arrow). Diffuse meningeal enhancement and a discrete rim of
enhancement ­surrounding the fusiform structure are present.
­monocytoid cells, large reactive lymphocytes, and rare eo-
sinophils also were observed. Interpretation: Marked neu-
trophilic pleocytosis (purulent inflammation).
Cytologic Analysis of CSF (Cisternal): A direct smear
of cisternal CSF contained disrupted cells and mildly to
moderately degenerate neutrophils. These were admixed
with low numbers of monocytoid cells and intermediate-
sized reactive lymphocytes. Rare neutrophils were observed
with intracellular rod-shaped bacteria. Few free rod-shaped
bacteria were noted in the background.
Microbiologic Testing: Aerobic urine culture: No growth
was detected.
Aerobic and anaerobic blood cultures: No growth was detected.
Aerobic and anaerobic CSF culture: Small numbers of Bacteroi-
des fragilis, positive for β-lactamase production.
Diagnosis: Bacteroides fragilis meningitis with extradural
abscess formation
Treatment and Outcome: Cocoa was treated with ticarcillin
(50 mg/kg IV q6h), metronidazole (10 mg/kg IV q8h), and
crystalloid fluids (LRS with 20 mEq/L KCl at 50 mL/h, IV).
The following day her mentation improved and she became
more willing to move. On day 3 after starting antibiotics,
SUGGESTED READING
Jang SS, Breher JE, Dabaco LA, et al. Organisms isolated from dogs and
cats with anaerobic infections and susceptibility to selected antimicro-
bial agents. J Am Vet Med Assoc. 1997;210:1610-1614.
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mg/kg PO q8h) and metronidazole (16 mg/kg PO q12h).
On day 4, Cocoa was less ataxic and had normal placing
reactions, but had a seizure while on a walk outside. The
seizure lasted 45 seconds and was manifested by jaw
chomping and defecation, but Cocoa did not fall over or lose
consciousness. Potassium bromide (40 mg/kg PO q8h for
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tionship between causative organisms and clinical characteristics of
infective endocarditis in dogs: 71 cases (1992-2005). J Am Vet Med
Assoc. 2006;228:1723-1734.
CHAPTER 38
Bordetellosis
Jane E. Sykes
Overview of Canine and Feline Bordetellosis
First Described: 1911 (Bacillus bronchicanis, Ferry).1 The genus
Bordetella was named after Bordet, who first described
Bordetella pertussis (Bordet and Gengou, 1906).2
Cause: Bordetella bronchiseptica, an aerobic gram-negative
motile bacterium (family Alcaligenaceae)
Affected Hosts: Dogs, cats, wild carnivores, pigs, horses, rab-
bits, rodents, turkey, humans. Dogs and cats share the
same strains.
Geographic Distribution: Worldwide
Mode of Transmission: Aerosol, contact with contaminated
fomites and water sources
Major Clinical Signs: Sneezing, serous to mucopurulent nasal
discharge, harsh or honking cough (especially dogs). Dogs
and cats with bronchopneumonia can develop fever,
lethargy, inappetence, tachypnea, productive cough, and
mucopurulent nasal and ocular discharge.
Differential Diagnoses: Upper and lower respiratory diseases
such as collapsing trachea, viral respiratory infections,
fungal pneumonia, protozoal pneumonia, respiratory
tract neoplasia, airway foreign bodies, chronic bronchitis,
eosinophilic bronchopneumopathy or granulomatosis,
parasitic infections (such as Filaroides, Oslerus, Capillaria,
Paragonimus, Dirofilaria), bronchopneumonias secondary
to conditions such as laryngeal paralysis or ciliary dyskine-
sia, left-sided congestive heart failure
Human Health Significance: B. bronchiseptica is closely
related to B. pertussis and Bordetella parapertussis, which
cause whooping cough in humans. Uncommonly, it has
been associated with respiratory disease in humans and
rarely systemic infections such as peritonitis, septicemia,
and meningitis. It primarily causes disease in the immu-
nocompromised. Contact with dogs and cats has been
associated with human infections, but host specificity
may also be strain related.
Etiologic Agent and Epidemiology
Bordetella spp. are small, pleomorphic, gram-negative cocco-
bacilli. They have fimbriae (pili) and can be motile by means
of flagella. There are at least nine different bacterial species in
the genus Bordetella (Table 38-1).3 The only species known
to cause disease in dogs and cats is Bordetella bronchiseptica,
372
but B. bronchiseptica is closely related to B. pertussis and
B. ­parapertussis, which cause pertussis (whooping cough) in
humans. In fact, there is evidence that B. pertussis evolved
from a distinct, human-associated lineage of B. bronchiseptica,4
and it has been recommended that B. bronchiseptica, B. per-
tussis, and B. parapertussis be reclassified as subspecies of the
“B. bronchiseptica cluster.”5 B. pertussis can colonize puppies
in the absence of clinical signs of respiratory disease.6
Worldwide, B. bronchiseptica is an important cause of respi-
ratory disease in dogs and cats; it also infects and causes respi-
ratory illness in many different animal species including wild
carnivores, pigs, rabbits, and occasionally horses, other herbi-
vores, rodents, turkeys, and humans. There are a large number
of different strains of B. bronchiseptica that vary in virulence
and host specificity. Nevertheless, the results of molecular typ-
ing efforts have shown that strains that infect dogs can be passed
to cats and vice versa.7-9 The whole genome of B. bronchiseptica
has been sequenced.10 Its genome is larger than that of its close
relatives, and it possesses several plasmids, some of which medi-
ate antimicrobial drug resistance.11
Like viral respiratory infections, bordetellosis is especially
prevalent in dogs and cats in shelter, pet stores, boarding
facilities, and other situations where large numbers of poten-
tially stressed animals may have been in close contact with one
another. Infections with B. bronchiseptica frequently occur in
concert with respiratory viral and/or Mycoplasma spp. infec-
tions (see Chapters 17, 23, and 40). Unlike B. pertussis and
B. parapertussis, B. bronchiseptica can persist in the envi-
ronment for at least 10 days and can grow in natural water
sources,12 but is susceptible to most disinfectants provided they
are used correctly.
The prevalence of B. bronchiseptica infections in group-
housed animals, including shelters, varies from group to
group, even in the same geographic region. B. bronchiseptica
can be isolated from apparently healthy cats and cats with
respiratory disease, but is more likely to be isolated from cats
with respiratory disease. In one European study that included
1748 cats from private multicat households, shelters, and
breeding catteries, the more cats in the group, the greater the
chance that B. bronchiseptica would be detected. In rescue
shelters, increased seroprevalence has been associated with
poor hygiene.13 In a study of 742 cats in the United Kingdom,
B. bronchiseptica was isolated from 0% of pet cats, 19.5% of
cats from rescue shelters, and 13.5% of cats from research col-
onies.14 B. bronchiseptica was cultured from 3.1% of 614 cats
from four shelters in Louisiana,15 5.1% of nasal swabs from
59 cats with acute respiratory disease in Colorado,16 and none
of 22 cats with respiratory disease in a shelter in California.17
However, the prevalence of infection was nearly 50% in nasal
 CHAPTER 38  Bordetellosis 373

TABLE 38-1
Species That Belong to the Genus Bordetella
Species Host(s) Disease
Bordetella Dogs, cats, pigs, Rhinitis, tracheobron-
bronchiseptica horses, rabbits, chitis, bronchopneu-
rodents, humans monia
Bordetella Humans Whooping cough
pertussis
Bordetella Humans, sheep Whooping cough
parapertussis (humans); ovine-
adapted strains
cause respiratory
disease in sheep
Bordetella Humans Upper respiratory tract FIGURE 38-1  Bordetella bronchiseptica infection. B. bronchiseptica is inhaled, adheres
holmesii disease, bacteremia to respiratory cilia by means of fimbrial adhesins, evades the immune system and secretes
Bordetella Humans Otitis media and a variety of toxins that damage the respiratory epithelium.
trematum wound infections
Bordetella avium Poultry (especially Rhinotracheitis and secretes toxins that damage the respiratory epithelium
turkey) (Figure 38-1). Adhesion of virulent strains of B. bronchiseptica
Bordetella hinzii Birds, rarely Normal flora of birds, to respiratory cilia occurs by means of fimbrial adhesins
humans bacteremia, cholan- (FIM), a filamentous hemagglutinin (FHA), pertactin, and
gitis in humans cell wall lipopolysaccharide. Bordetella colonization factor
A (BcfA) is another outer membrane protein that appears to
Bordetella petrii Environment, None, osteomyelitis be important for tracheal colonization by B. bronchiseptica.21
rarely humans B. bronchiseptica possesses genes that encode for pertussis
Bordetella Humans Bacteremia, skin infec- toxin, an important adhesin in B. pertussis. These genes are
ansorpii tion not expressed in vitro, but there is some evidence for pertussis
toxin expression in vivo.22 Evasion of host defenses is mediated
by the organism’s outer capsule (which is absent in B. pertus-
swabs from 40 cats with rhinitis from a shelter in Colorado.18 sis and B. parapertussis), adenylate cyclase toxin, and pertac-
Similarly, a high prevalence (26%) of infection was detected tin. The capsule protects it from phagocytosis and destruction
with a PCR assay in 100 cats with upper respiratory disease by complement. Adenylate cyclase toxin catalyzes the con-
from central Italy.19 version of ATP to cAMP, which inhibits the migration and
Only a few studies have examined the epidemiology of activation of phagocytes and T-lymphocytes. Pertactin allows
B. bronchiseptica infection in dogs. In a case review from the the organism to resist neutrophil-mediated clearance.23 A type
eastern United States, B. bronchiseptica was isolated from VI secretion system also appears to play a key role in per-
32 (49%) of 65 pet dogs under 1 year of age with radiographic sistence of B. bronchiseptica.24 Other virulence determinants
and microbiologic evidence of bacterial bronchopneumonia. present in the outer membrane include lipopolysaccharide
Most of these dogs had originated from breeders (40%) or pet and the Brk protein (Bordetella resistance to killing), which
stores (41%), and only 8% were from shelters. Dogs infected allow the organism to resist complement-mediated destruc-
with B. bronchiseptica were younger (7 to 35 weeks of age), had tion. At least two other important exotoxins damage respira-
been owned for a shorter period of time (median, 18 days), were tory epithelial cells and cells of the immune system, and incite
more likely to have originated from a pet store and were less cytokine production: tracheal cytotoxin (TCT) and dermone-
likely to have originated from a breeder than dogs with pneu- crotic toxin (DNT). B. bronchiseptica also possesses a type
monia caused by other organisms. B. bronchiseptica was iso- III secretion system that allows it to inject as-yet-undefined
lated from dogs with respiratory disease as well as from healthy effector proteins into cellular targets.5
dogs in a rehoming kennel in the United Kingdom.20 The inflammation and altered cell function that occur as
a result of B. bronchiseptica infection lead to increased fluid
Clinical Features and mucus secretion, and impairment in host innate immune
defenses predisposes to opportunistic viral and bacterial infec-
Signs and Their Pathogenesis tions. Clinical signs vary in severity and may reflect factors
Transmission of B. bronchiseptica occurs primarily by the such as the bacterial strain involved, host immunity, and the
airborne route, but contact with contaminated fomites and presence of co-infections. The incubation period ranges from
water sources may also be important. The organism is highly 2 to 10 days. Rhinitis and tracheobronchitis may be associated
contagious. Much of what is known about B. bronchiseptica with serous to mucopurulent nasal discharge, sneezing, stertor,
pathogenesis is based on studies of infections in host spe- and, especially in dogs, a persistent, often paroxysmal harsh
cies other than the dog or cat. B. bronchiseptica is inhaled, cough. In some dogs, development of bronchopneumonia leads
adheres to respiratory cilia, evades the immune system, to fever, productive cough, lethargy, and decreased appetite.
374 SECTION 2  Bacterial Diseases
FIGURE 38-2  Kitten with Bordetella bronchiseptica conjunctivitis and bronchopneu-
monia. The kitten was tachypneic and had mucopurulent ocular discharge.
Bronchopneumonia may be due to B. bronchiseptica itself or
it may result from co-infection with other respiratory patho-
gens, such as canine distemper virus (CDV). In cats, infection
is significantly associated with sneezing, and cough is uncom-
mon.14 Cats with pneumonia may develop tachypnea, cyanosis,
and death.
Shedding of B. bronchiseptica by both dogs and cats can con-
tinue intermittently for at least a month and sometimes several
months after infection. Evasion of the immune system through
survival inside phagocytes may explain the persistent infection
that occurs. In contrast, B. pertussis is rapidly destroyed by
macrophages.25
Physical Examination Findings
On physical examination, dogs with uncomplicated borde-
tellosis are bright, alert, and active with a paroxysmal cough
that can occur throughout the examination. The cough is
often easily elicited on tracheal or laryngeal palpation. Con-
junctivitis and serous to mucopurulent ocular and nasal dis-
charge may be present in affected dogs and cats (Figure 38-2).
Dogs and cats with complicated Bordetella bronchopneumo-
nia may be pyrexic, lethargic, and tachypneic with increased
respiratory effort. There may also be increased lung sounds
and/or referred upper airway noises on thoracic auscultation,
and mucopurulent nasal discharge.
Diagnosis
Laboratory Abnormalities
There are no specific laboratory abnormalities in uncompli-
cated bordetellosis. Usually the CBC is normal. Dogs with
bronchopneumonia can also have a normal CBC,26 or a mild
to moderate neutrophilia, bandemia, and toxic neutrophils
may be present. Cytologic examination of transtracheal
lavage (TTL) or bronchoalveolar lavage (BAL) specimens
may reveal a suppurative or mixed exudate, and some-
times intracellular and extracellular coccobacilli are visible
(Figure 38-3).
FIGURE 38-3  Bronchoalveolar lavage cytology from a dog infected with Bordetella
bronchiseptica. Numerous coccobacilli are adhered to the cilia of columnar epithelial cells.
Wright stain, 1000× oil magnification. (Courtesy of Michael Scott, Michigan State Univer-
sity. In Raskin RE, Meyer D, eds. Canine and Feline Cytology: A Color Atlas and Interpretative
Guide, 2 ed, St. Louis, MO: Saunders; 2010.)
Diagnostic Imaging
Plain Radiography
Plain thoracic radiographs in dogs with uncomplicated borde-
tellosis may be unremarkable or show a mild diffuse interstitial
or bronchointerstitial pattern. Bordetella bronchopneumonia
may be characterized by development of peribronchial and alve-
olar infiltrates and lobar consolidation.
Microbiologic Tests
The main assays used for diagnosis of bordetellosis in dogs and cats
are aerobic bacterial culture and PCR assays, which can be per-
formed on nasal or oropharyngeal swabs, TTL or BAL specimens,
or respiratory tract tissue collected at necropsy (Table 38-2). In
some studies, B. bronchiseptica has been detected in nasal but not
oropharyngeal swabs, but in other studies, the use of oropharyn-
geal swabs was more sensitive. Collection of swab specimens from
the nasal cavity or posterior nasopharynx is preferred for diagnosis
of pertussis in humans because these regions have ciliated epithelial
cells, for which B. pertussis has affinity.27 False-negative results
occur when organism numbers in secretions are low, and depend-
ing on the sensitivity of the PCR assay used and the viability of the
bacteria in the specimen, culture may be positive when PCR is neg-
ative and vice versa. Although the detection of B. bronchiseptica
in TTL or BAL specimens is diagnostic for bordetellosis, it may
be more difficult to interpret the significance of a positive culture
or PCR assay result from nasal or oropharyngeal swabs, espe-
cially among group-housed dogs and cats when the background
prevalence of infection is high. Co-infections with viral respiratory
pathogens should always be considered when B. bronchiseptica is
detected, as well as the presence of other comorbidities that sup-
press the innate or adaptive immune system that could predispose
to bordetellosis (e.g., ciliary dyskinesia or brachycephalic obstruc-
tive syndrome in dogs, retroviral infections in cats).
Bacterial Culture
B. bronchiseptica can usually be isolated on routine aerobic bac-
terial culture media, such as MacConkey agar and blood agar.

TABLE 38-2
Diagnostic Assays Available for Bordetellosis in Dogs and Cats
Assay Specimen Type Target
Aerobic bacterial Nasal and oropharyngeal B. bronchiseptica
culture swabs, transtracheal and
bronchoalveolar lavage
specimens, airway and
lung specimens collected
at necropsy
PCR assays See Aerobic bacterial B. bronchiseptica
culture DNA
Serology Serum Antibodies against
Bordetella antigens
Charcoal-based medium that is supplemented with cephalexin is
preferred for isolation of B. pertussis, which is more fastidious.
For B. bronchiseptica, use of methicillin or oxacillin in selective
media can prevent overgrowth by contaminating microflora.5
The laboratory should be informed when bordetellosis is on
the differential diagnosis list. In one study, B. bronchiseptica
was isolated from cats using nasal swabs but not oropharyngeal
swabs.16 The use of Dacron, rayon, or calcium alginate swabs is
preferred for culture of B. pertussis, because growth is inhibited
when cotton swabs are used.27 Unlike PCR assays, successful
detection of B. bronchiseptica using culture allows susceptibility
testing, which assists in rational antimicrobial drug selection.
Molecular Diagnosis Using the Polymerase Chain Reaction
Sensitive and specific real-time PCR assays that detect
B. bronchiseptica DNA have been described,13,28 and assays are
available through several commercial veterinary diagnostic labo-
ratories. Some laboratories offer panels that also detect other
respiratory pathogens, which can be useful for detection of coin-
fections. As with culture, negative results can occur when organ-
ism numbers are low in the specimen, or when there has been
recent antimicrobial drug use; however, PCR assays may be more
likely to be positive than culture in these situations. Positive PCR
assay results can occur for at least 3 weeks after intranasal vac-
cination.29 A conventional PCR assay that differentiates between
field strains and one vaccine strain of B. bronchiseptica has been
described,29 but commercially available real-time assays that dis-
tinguish between field and vaccine strains are needed.
Serologic Diagnosis
Antibodies to B. bronchiseptica can be detected using bacte-
rial agglutination or recombinant or whole-cell ELISA assays,
but serology is of limited use for diagnosis because of the high
prevalence of antibodies in the dog and cat population and the
influence of vaccination, which is routinely performed on entry
CHAPTER 38  Bordetellosis 375
Performance
False negatives can occur when specimens contain no
or low numbers of organisms, or after antimicrobial
treatment. Allows subsequent susceptibility testing.
The clinical significance of positive culture results
from nasal and oropharyngeal swab specimens may be
difficult to interpret.
Sensitivity and specificity can vary depending on assay
design. False negatives can occur when specimens
contain no or low numbers of organisms, or after
antimicrobial treatment. Testing specimens from
multiple different anatomic sites or combining PCR
assays with other diagnostic tests can increase sensitiv-
ity. Avirulent live vaccine organisms may be detected
for several weeks after intranasal vaccination. Because
of subclinical shedding, the clinical significance of a
positive result may be difficult to interpret.
Interpretation complicated by previous vaccination
and widespread exposure.
to most shelters. Serology has primarily been used to study the
epidemiology of infection and to examine immune responses as
they relate to vaccination.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with uncomplicated borde-
tellosis are usually minimal. There may be an accumulation of
mucopurulent exudate in the airways.
Histopathologic Findings
Microscopic findings in dogs or cats with bordetellosis consist
of a suppurative inflammatory response that is usually confined
to the ciliated portions of the respiratory tract. Cilia are mostly
intact, but regional bronchial epithelial necrosis may be present.
Mild lymphoid hyperplasia may be present. Depending on the
stage and severity of infection, aggregates of bacteria may be
observed in association with the cilia.30
Treatment and Prognosis
Antimicrobial Treatment
B. bronchiseptica isolates from dogs and cats have the propen-
sity to exhibit multidrug resistance (i.e., resistance to three or
more antimicrobial drug classes). Resistance to amoxicillin and
trimethoprim-sulfamethoxazole is common. Many infections
are mild or self-limiting, and so antimicrobial drug treatment
should be reserved for animals with confirmed B. bronchiseptica
infection that have persistent clinical signs (>7 to 10 days) or
those with more severe clinical signs and radiographic abnor-
malities that suggest bronchopneumonia. Young kittens and
puppies (<6 to 8 weeks of age) are candidates for early initiation
of treatment because disease in neonates may progress rapidly
to pneumonia and death. Treatment should be based when-
ever possible on the results of culture and susceptibility testing.
376 SECTION 2  Bacterial Diseases
When susceptibility results are not available, or when they are
pending and bordetellosis is strongly suspected, the initial drug
of choice is doxycycline. At the time of writing, doxycycline
resistance is uncommon among isolates from dogs and cats,31-33
and doxycycline effectively penetrates lung tissue. Unfortu-
nately, doxycycline is not the best choice for other secondary
bacterial pneumonias because it is bacteriostatic and parenteral
doxycycline can be expensive and difficult to administer. This
reinforces the need for culture whenever feasible in animals
with evidence of pneumonia. Bacterial shedding may continue
despite resolution of clinical signs, so affected dogs and cats
should remain in isolation from other animals whenever pos-
sible for at least 2 months after treatment.
Rarely, treatment with systemic antimicrobial drugs fails to
resolve clinical signs of bordetellosis, and the organism persists
despite documented susceptibility to the antimicrobial drug
selected. In this situation, a short period of nebulized amino-
glycosides could be considered. Because this can cause airway
irritation, its use should probably be restricted for dogs with
refractory bronchopneumonia, or severe and persistent tracheo-
bronchitis (for example, >4 weeks in duration).
Supportive Care and Prognosis
The prognosis for dogs with uncomplicated bordetellosis is
excellent. Cats and dogs with bronchopneumonia usually
require other supportive treatments, such as IV fluid therapy,
appropriate nutritional support, supplemental oxygen, and neb-
ulization (see Chapters 17 and 23). In one study, bronchopneu-
monia associated with B. bronchiseptica infection was more
severe than that associated with other bacterial agents, and
affected dogs also had higher PvCO2 values, were more likely to
require supplemental oxygen, and had longer mean hospitaliza-
tion times (7.2 days compared with 4.9 days).26
Immunity and Vaccination
Antibodies to outer surface molecules of B. bronchiseptica,
especially IgA in mucosal secretions, are important for bacterial
clearance. Several avirulent live, mucosal (intranasal or oral)
B. bronchiseptica vaccines are available for dogs, some of which
also contain canine parainfluenza virus with or without canine
adenovirus 2. These vaccines are designed to stimulate mucosal
immunity and provide protection within 3 days after a single
dose of vaccine.34 Protection with intranasal vaccines also occurs
in the face of maternal antibody and can last at least 1 year.35,36
Inactivated parenteral B. bronchiseptica vaccines are also avail-
able for dogs, but two doses administered 3 to 4 weeks apart are
required to stimulate maximal immunity, which occurs 1 week
after the second dose. The rapid onset of protection that fol-
lows the use of intranasal vaccines makes them the vaccine type
of choice for puppies and animals introduced into heavily con-
taminated environments, such as shelters, when immunization
in advance of entry is usually not possible. Mucosal vaccines
should not be given to animals treated with antimicrobial drugs
(because they inactivate bacteria in the vaccine), and admin-
istration of intranasal vaccines to aggressive animals without
sedation may not be feasible. Transient signs of mild to moder-
ate respiratory disease occur in a small percentage of vaccinated
dogs and cats several days after mucosal vaccination. In shel-
ters, it may not be possible to differentiate between postvaccinal
disease and infectious respiratory disease due to field pathogens,
which complicates decisions that relate to isolation practices.
Inadvertent parenteral administration of intranasal vaccines has
the potential to lead to injection site reactions, hepatic necrosis,
and even death in some dogs.37 Subcutaneous fluids should be
administered at the site of vaccination, and immediate treatment
with antimicrobial drugs is indicated. It has been suggested that
gentamicin sulfate solution be instilled into the affected area
(2 to 4 mg/kg gentamicin sulfate in 10 to 30 mL of saline),
and dogs should then be treated with oral doxycycline for
5 to 7 days.38 Special packaging has been developed by some
vaccine manufacturers in an attempt to prevent inadvertent par-
enteral administration of the intranasal vaccine.
Concerns have been raised that intranasal B. bronchisep-
tica vaccines may cause respiratory disease in immunosup-
pressed humans who inhale the vaccine during administration
or who contact vaccine organisms shed by recently immunized
dogs.39,40 However, as yet, there has been no molecular proof
that the B. bronchiseptica strains isolated from affected human
patients match vaccine strains. In fact, in one report of B. bron-
chiseptica pneumonia in an infant that was temporally related
to intranasal vaccination of a pet dog, genetic analysis (ribotyp-
ing) showed that the vaccine was not the source of the infant’s
exposure.41 The organism matched to another B. bronchiseptica
strain that had previously been isolated from a human patient.
Intranasal B. bronchiseptica vaccination is followed by the
development of low titers of serum IgG, whereas serum IgG
responses are higher after parenteral vaccination (as might be
expected). Parenteral whole-cell vaccines, and more recently
acellular vaccines that include virulence factors such as pertac-
tin, FIM, FHA, and pertussis toxin, are widely used to prevent
pertussis in humans.26 Fewer scientific data are available on the
efficacy of parenteral B. bronchiseptica vaccines in dogs when
compared with intranasal vaccines. One study showed that an
intranasal vaccine was more effective than a parenteral anti-
gen extract vaccine.42 Additional challenge studies are required
that directly compare the efficacy of intranasal and parenteral
B. bronchiseptica vaccines for dogs.
In some countries, intranasal B. bronchiseptica vaccines are
available for cats, which can reduce signs of disease after chal-
lenge as long as 1 year after vaccination.43 Because disease is
rarely severe in cats more than 6 weeks of age, vaccination of
cats has not been recommended on a routine basis,43 but could
be considered as an adjunct to management strategies such as
reduction of overcrowding and proper disinfection if outbreaks
of bordetellosis are a problem in group-housed cats. Vaccina-
tion of immunocompromised cats is not recommended.44
Prevention
In the absence of management strategies to reduce stress, over-
crowding, and comorbidities, vaccination is unlikely to com-
pletely prevent bordetellosis. The reader is referred to the
sections on prevention in Chapters 17 (dogs) and 23 (cats) for
more additional information on prevention and management of
outbreaks of respiratory infections in group-housed animals.
Public Health Aspects
B. bronchiseptica is an uncommon cause of respiratory disease
in humans, but has been isolated from humans without clini-
cal signs and humans with mild to severe respiratory disease,
including pneumonia.45 Most, but not all, affected humans have
underlying immunosuppressive conditions that predispose them

to infection. B. bronchiseptica peritonitis has been reported in
human patients receiving continuous peritoneal dialysis.46,47
Bacteremia and meningitis have also been reported.48 Recent
exposure to cats and to dogs with signs of “kennel cough” has
been noted in most human cases,45,49 but airborne transmis-
sion between humans can occur in hospital settings.50 At least
in mouse models, human acellular pertussis vaccines appear
to cross-protect against B. bronchiseptica.51 Infections with
B. bronchiseptica may be underrecognized in people, because the
direct fluorescent antibody test used for diagnosis of pertussis
CASE EXAMPLE
Signalment: 7-week-old intact female Persian kitten from
California
History: A research colony kitten was examined for a 3-day
history of respiratory signs. Three days previously, sneezing
was observed in this kitten and one of its littermates. A
day later, the kitten developed lethargy, inappetence, and
respiratory distress, and the littermate was found dead. The
kitten had been treated with lactated Ringer’s solution (6
mL SC q24h) and azithromycin suspension (0.7 mg/kg PO
q24h) since it became unwell. Two other kittens in the litter
remained unaffected, and one had mild nasal discharge.
All of the cats had tested negative for FeLV antigen and FIV
antibody using a commercially available in-house ELISA kit.
Physical Examination:
Body Weight: 0.37 kg
General: Alert, open-mouth breathing. T = 97.5°F (36.4°C),
HR = 220 beats/min, RR = 40 breaths/min, mucous
membranes pale pink.
Eyes, Ears, Nose, and Throat: No abnormalities were noted.
Musculoskeletal: Body condition score 4/9.
Respiratory: Markedly increased inspiratory and expiratory
effort, tachypnea, and open-mouth breathing were noted.
Increased lung sounds were detected on auscultation.
Cardiovascular, Gastrointestinal, and Genitourinary
Systems and Lymph Nodes: No clinically significant
abnormalities were detected.
Imaging Findings: Thoracic radiographs: There was collapse
of the left cranial lung lobe and partial volume loss in the
left caudal lung lobe with evidence of mediastinal shift to
the left, possibly secondary to a bronchial mucus plug. A
focal increase in opacity was noted in the area of the right
middle lung lobe with hyperinflation of the right cranial and
caudal lung lobes. There were regions of gas trapping, and
the bronchi were enlarged. Emphysema was suspected,
SUGGESTED READINGS
Berkelman RL. Human illness associated with use of veterinary vac-
cines. Clin Infect Dis. 2003;37:407-414.
Mattoo S, Cherry JD. Molecular pathogenesis, epidemiology, and clinical
manifestations of respiratory infections due to Bordetella pertussis and
other Bordetella subspecies. Clin Microbiol Rev. 2005;18:326-382.
CHAPTER 38  Bordetellosis 377
may not distinguish between B. pertussis and B. bronchiseptica.
Clinically, recognition of B. bronchiseptica infections in humans
is important, because B. bronchiseptica is resistant to macrolides,
which are first-line agents for pertussis.41 Until more is under-
stood about the risks of canine mucosal B. bronchiseptica vac-
cines for immunocompromised people, parenteral vaccination of
at-risk dogs owned by seriously immunocompromised humans
is probably warranted, and the owners of these dogs should be
instructed to avoid or minimize situations in which their dogs
could come into contact with large numbers of other dogs.
possibly congenital. Consolidation in the area of the right
middle lung lobe was compatible with pneumonia.
Outcome: The kitten was treated with oxygen supplementation,
terbutaline (0.01 mg/kg SC q6h), ticarcillin-clavulanate (50
mg/kg IV q8h), LRS with 5% dextrose (1 mL/hr IV), a single
dose of dexamethasone (1 mg/kg IV), and a single dose of
furosemide (2 mg/kg IV). Euthanasia was performed 24 hours
later due to lack of improvement and the poor prognosis.
Necropsy Findings: An endotracheal wash was performed
immediately after euthanasia. This revealed marked, septic,
suppurative inflammation and mild to moderate epithelial
hyperplasia. Bacteria were present in large numbers
extracellularly, within degenerate neutrophils, and in large mats.
Gross necropsy findings consisted of diffuse lung lobe collapse
with multiple firm dark red areas throughout the paren-
chyma. On histopathology, there was severe, multi-focal
to coalescing, acute necrosuppurative bronchopneumonia
with abundant clusters of gram-negative bacteria. Exami-
nation of the upper respiratory tract showed mild, multifo-
cal neutrophilic rhinitis and mild, multifocal, lymphocytic
tracheitis. The other kitten was also necropsied, with similar
findings.
Microbiologic Testing (Endotracheal Wash Specimen):
Culture for aerobic bacteria, Mycoplasma spp., and virus isolation
revealed only moderate numbers of Bordetella bronchiseptica.
Diagnosis: Bordetella bronchiseptica bronchopneumonia.
Comments: This outbreak of severe bordetellosis in a research
cat colony resulted in the death of several kittens, which
deteriorated rapidly even in the face of supportive care.
Co-infections were not detected, although PCR testing
was not performed and the negative Mycoplasma culture
and virus isolation results did not rule out the possibility
of co-infection. Although susceptibility testing was not
performed, resistance to the antimicrobials that were used
can occur in B. bronchiseptica. Furthermore, the dose of
azithromycin used as reported in the medical record was
subtherapeutic.
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378 SECTION 2  Bacterial Diseases
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septica in cats: experience from 218 European catteries. Vet Rec.
2005;156:669-673.
14. Binns SH, Dawson S, Speakman AJ, et  al. Prevalence and risk
factors for feline Bordetella bronchiseptica infection. Vet Rec.
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15. Hoskins JD, Williams J, Roy AF, et al. Isolation and characteriza-
tion of Bordetella bronchiseptica from cats in southern Louisiana.
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16. Veir JK, Ruch-Gallie R, Spindel ME, et al. Prevalence of selected
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17. Burns RE, Wagner DC, Leutenegger CM, et  al. Histologic and
molecular correlation in shelter cats with acute upper respiratory
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18. Spindel ME, Veir JK, Radecki SV, et  al. Evaluation of prado-
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19. Di Martino B, Di Francesco CE, Meridiani I, et al. Etiological inves-
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20. Chalker VJ, Toomey C, Opperman S, et  al. Respiratory disease
in kennelled dogs: serological responses to Bordetella bronchi-
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22. Stefanelli P, Mastrantonio P, Hausman SZ, et al. Molecular char-
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23. Inatsuka CS, Xu Q, Vujkovic-Cvijin I, et al. Pertactin is required
for Bordetella species to resist neutrophil-mediated clearance.
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24. Weyrich LS, Rolin OY, Muse SJ, et al. A type VI secretion system
encoding locus is required for Bordetella bronchiseptica immuno-
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opposite effects on intracellular survival of Bordetella pertussis
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26. Radhakrishnan A, Drobatz KJ, Culp WT, et  al. Community-
acquired infectious pneumonia in puppies: 65 cases (1993-2002).
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29. Iemura R, Tsukatani R, Micallef MJ, et al. Simultaneous analysis of
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31. Speakman AJ, Binns SH, Dawson S, et al. Antimicrobial susceptibility
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the agar dilution and E-test methods. Vet Microbiol. 1997;54:63-72.
32. Speakman AJ, Dawson S, Corkill JE, et al. Antibiotic susceptibil-
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39. Gisel JJ, Brumble LM, Johnson MM. Bordetella bronchiseptica
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42. Davis R, Jayappa H, Abdelmagid OY, et  al. Comparison of the
mucosal immune response in dogs vaccinated with either an intrana-
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novel intranasal vaccine against feline bordetellosis. Vet Rec.
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infection in cats. ABCD guidelines on prevention and management.
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of confirmed Bordetella bronchiseptica infection and colonization
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CHAPTER 38  Bordetellosis 379
49. Goldberg JD, Kamboj M, Ford R, et al. “Kennel cough” in a patient
following allogeneic hematopoietic stem cell transplant. Bone Mar-
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Bordetella bronchiseptica infection following hematopoietic stem cell
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51. Goebel EM, Zhang X, Harvill ET. Bordetella pertussis infection or
vaccination substantially protects mice against B. bronchiseptica
infection. PLoS One. 2009;4:e6778.
CHAPTER 39

Cell Wall–Deficient Bacterial Infections

Jane E. Sykes

Clinical Features
Overview of Cell Wall–Deficient Bacterial
Infections In human patients, CWDB have been implicated in culture-­
First Described: 1935, London (Kleineberger)1 negative endocarditis, bacteremia, uveitis, pneumonia, osteomy-
elitis and arthritis, urinary tract infections, soft tissue infections,
Causes: Cell wall–deficient strains of a variety of gram-positive
and meningitis.3 In cats, CWDB have been implicated as a cause
and gram-negative bacteria
of subcutaneous abscesses and arthritis.4,5 A cell wall–deficient
Geographic Distribution: Worldwide Nocardia sp. was isolated from a dog with polyarthritis that
Major Clinical Signs: Chronic draining or ulcerated soft tissue was refractory to antibiotics,6 and a Pseudomonas aeruginosa
and skin lesions, polyarthritis, possibly other chronic pyo- L-form was isolated from the blood of a dog after antibiotic
genic and pyogranulomatous lesions treatment for P. aeruginosa endocarditis.7
Differential Diagnoses: Infections due to other fastidious
bacteria such as Mycoplasma, Nocardia, Mycobacterium,
Diagnosis
Bartonella, Brucella, nutritionally variant streptococci such Physical examination and laboratory findings in cats and dogs
as Granulicatella, or, for animals with polyarthritis, Bor- infected with CWDB may be similar to those for other chronic,
relia burgdorferi; fungal infections (e.g., due to Sporothrix persistent bacterial infections. Infection with CWDB should be
schenckii or Coccidioides immitis); persistence of a foreign considered in cats and dogs with culture-negative pyogranulo-
body; neoplasia; congenital immunodeficiency; primary matous or suppurative inflammatory lesions, or when a bacte-
immune-mediated disease rial etiology is suspected on the basis of clinical signs but routine
Human Health Significance: Likely similar to that for the cor-
responding walled (or “normal”) variant of the bacterial
species involved
BOX 39-1
Examples of Medically Important Bacteria
That May Become Cell Wall–Deficient When Subjected
to Appropriate Stressors
Etiologic Agent and Epidemiology
Staphylococcus spp.
Cell wall–deficient bacteria (CWDB), also known as L-phase Streptococcus spp.
or L-form bacteria, are bacterial variants that lack a cell wall, Enterococcus spp.
although they may in fact possess small amounts of peptido- Escherichia coli
glycan.2 The name L-form was given to these bacteria because Serratia marcescens
they were discovered at the Lister Institute in London. L-form Pasteurella multocida
bacteria are distinct from mycoplasmas, because Mycoplasma Pseudomonas aeruginosa
spp. do not originate from bacteria that normally possess a Proteus mirabilis
cell wall. A huge variety of gram-­positive and gram-negative Salmonella typhimurium
bacterial species may become CWDB when exposed to cer- Lactobacillus spp.
tain stressors in the laboratory (such as antimicrobial drugs) Nocardia spp.
(Box 39-1). Some of these bacteria remain as CWDB (stable Actinomyces spp.
L-forms), whereas others revert back to possession of a cell Bacillus spp.
wall (unstable L-forms). CWDB assume a spherical or pleo- Brucella spp.
morphic shape, and are susceptible to osmotic lysis. How- Corynebacterium spp.
ever, they resist β-lactam drugs such as penicillin, and may be Borrelia burgdorferi
able to evade the innate immune response. Thus, it has been Leptospira interrogans
proposed that reversion to cell wall–deficient forms may be a
mechanism of bacterial persistence. Although the formation Adapted from Onwuamaegbu ME, Belcher RA, Soare C. Cell wall-
of CWDB has been well documented in the laboratory, the deficient bacteria as a cause of infections: a review of the clinical
role that CWDB play in disease remains controversial.3 significance. J Int Med Res 2005;33:1-20.

380
 CHAPTER 39  Cell Wall–Deficient Bacterial Infections
TABLE 39-1
Assays Available for Diagnosis of Cell Wall–Deficient Bacterial Infections in Dogs and Cats
Assay Specimen Type Target
Culture Biopsies, body fluids, Cell wall–deficient
wash specimens bacteria (CWDB)
Cytology or Biopsies, smears of CWDB
­histopathology affected tissues
Electron microscopy Biopsies CWDB
Broad-range bacte- Biopsies, body fluids, 16S ribosomal subunit
rial PCR assay wash specimens DNA of CWDB
and sequencing
cultures are negative. Definitive diagnosis is difficult because the
organisms are not always readily visualized using light micros-
copy, and passage through special hypertonic laboratory media
is required for growth of CWDB in culture (Table 39-1). Elec-
tron microscopy has been used to identify the organisms in
lesions. The use of broad-range bacterial PCR assays that detect
bacterial 16S ribosomal subunit DNA in tissues may be use-
ful to detect L-form bacteria in tissues in the future when rou-
tine culture methods are negative, although this method does
not distinguish between CWDB and their walled counterparts.
Infection with CWDB could also be suspected when lesions
resolve after treatment with tetracyclines.
Treatment and Prognosis
CWDB are generally susceptible to tetracyclines such as
doxy­cycline. Other antimicrobial drugs that act through cell
wall–­independent mechanisms, such as fluoroquinolones, chlor-
amphenicol, and macrolides, may also be active against some
L-form bacteria.
Public Health Aspects
The role that CWDB play in zoonotic disease is unclear, but it
is reasonable to believe it is similar to that of the corresponding
walled (or normal) variant of the bacterial species implicated. A
single report described isolation of an L-form of Streptococcus
sanguinis from a vascular graft of a hemodialysis patient.8 The
affected person’s pet dog was reported to often lick the graft site,
and S. sanguinis was also isolated from the dog’s oral cavity.
Although this was suggestive of zoonotic transmission, the iden-
tity of the isolates was not confirmed with molecular methods.
381
Performance
Requires special L-form bacterial (hyperosmolar)
media
Organisms usually not visible owing to the lack of a
cell wall, but may be gram-variable. Low sensitiv-
ity and cannot be used to identify absence of a
cell wall (although the presence of organisms that
stain strongly with Gram stain indicates the pres-
ence of walled bacteria).
Confirms the presence of CWDB in lesions. May not
distinguish CWDB from Mycoplasma spp. Often
expensive and not readily available for routine
diagnostic purposes.
Confirms presence of bacteria in lesions when
culture is negative. False positives may occur if
contaminating bacteria are present. Does not
distinguish between CWDB and their walled
counterparts. Sensitivity and specificity may vary
between assays.
SUGGESTED READINGS
Carro T, Pedersen NC, Beaman BL, et al. Subcutaneous abscesses and
arthritis caused by a probable bacterial L-form in cats. J Am Vet Med
Assoc. 1989;194:1583-1588.
Chmel H. Graft infection and bacteremia with a tolerant L-form of
Streptococcus sanguis in a patient receiving hemodialysis. J Clin
Microbiol. 1986;24:294-295.
REFERENCES
1. Kleineberger E. The natural occurrence of pleuropneumonia-like
organisms in apparent symbiosis with Streptobacillus moniliformis
and other bacteria. J Pathol Bacteriol. 1935;40:93-105.
2. Joseleau-Petit D, Liebart JC, Ayala JA, et  al. Unstable Escherichia
coli L forms revisited: growth requires peptidoglycan synthesis.
J Bacteriol. 2007;189:6512-6520.
3. Onwuamaegbu ME, Belcher RA, Soare C. Cell wall–deficient bacte-
ria as a cause of infections: a review of the clinical significance. J Int
Med Res. 2005;33:1-20.
4. Carro T, Pedersen NC, Beaman BL, et  al. Subcutaneous abscesses
and arthritis caused by a probable bacterial L-form in cats. J Am Vet
Med Assoc. 1989;194:1583-1588.
5. Keane DP. Chronic abscesses in cats associated with an organism
resembling Mycoplasma. Can Vet J. 1983;24:289-291.
6. Buchanan AM, Beaman BL, Pedersen NC, et al. Nocardia asteroides
recovery from a dog with steroid- and antibiotic-unresponsive idio-
pathic polyarthritis. J Clin Microbiol. 1983;18:702-708.
7. Bone WJ. L-form of Pseudomonas aeruginosa the etiologic agent
of bacterial endocarditis in a dog. Vet Med Small Anim Clin.
1970;65:224-227.
8. Chmel H. Graft infection and bacteremia with a tolerant L-form
of Streptococcus sanguis in a patient receiving hemodialysis. J Clin
Microbiol. 1986;24:294-295.
CHAPTER 40

Mycoplasma Infections
Jane E. Sykes

Overview of Mycoplasma and Ureaplasma BOX 40-1

Infections Nonhemotropic Mycoplasma and Ureaplasma Species Found
First Described: Dogs, Japan 1934 (Shoetensack).1 Reports of
in Dogs and Cats3-7
human Mycoplasma infections also occurred around this
time.2
Dogs Cats
Causes: Mycoplasma cynos and Mycoplasma felis have been M. cynos M. felis
most commonly associated with disease, but many other M. canis M. gateae
Mycoplasma species infect dogs and cats M. molare M. arginini
Geographic Distribution: Worldwide M. arginini M. feliminutum
M. bovigenitalium M. canadense*
Mode of Transmission: Direct contact, fomite transmission
M. edwardii M. cynos*
in overcrowded environments. Opportunistic invasion of
M. feliminutum M. lipophilum*
commensal mycoplasmas also occurs.
M. felis M. hyopharyngis*
Major Clinical Signs: Conjunctivitis, sneezing and nasal dis- M. gateae U. felinum
charge (cats); cough, tachypnea, fever, weight loss (lower M. maculosum U. cati
respiratory disease); swollen, painful joints (polyarthritis); M. opalescens Acholeplasma laidlawii
subcutaneous abscesses; neurologic signs (meningoen- M. spumans
cephalitis); prostatitis, epididymitis-orchitis, or lower uri- M. sp. HRC689
nary tract signs (dogs) M. sp. VJC358
Differential Diagnoses: Primarily gram-positive and gram- U. canigenitalium
negative bacterial infections. Differential diagnoses for Acholeplasma laidlawii
suspected mycoplasma respiratory disease include viral
infections, fungal and parasitic causes of pneumonia, aspi- *Identified on the basis of DNA sequencing of small segments of the
ration pneumonia, and neoplasia. 16S rRNA gene; sequence analysis of longer fragments may reveal
Human Health Significance: Canine and feline mycoplasma these are in fact other mycoplasma species.
species have rarely been detected in immunocompro-
mised humans with mycoplasmosis who have had con-
successfully cultured in the laboratory (see Chapter 41). The
tact with pet cats and dogs.
remainder of this chapter refers only to nonhemotropic myco-
plasma species.
Mycoplasmas are commensal bacteria found widely in asso-
ciation with mucous membranes of all mammalian species.
Etiology and Epidemiology Simultaneous colonization with more than one Mycoplasma
species is common. Mycoplasmas have also been associated
Mycoplasmas are fastidious bacteria that lack a cell wall. They with keratoconjunctivitis and/or upper respiratory tract disease
belong to the class Mollicutes (which translates to “soft skin”), (URTD) in cats; reproductive disease and urinary tract infec-
and are the smallest known free-living organisms. Many require tions (UTIs) in dogs; and lower respiratory tract disease, skin
sterols for growth, and Ureaplasma species require urea for fer- and soft tissue infections, meningoencephalitis, and arthritis in
mentation. Mycoplasmas measure only 0.3 to 0.8 µm in size. both cats and dogs (Box 40-2).8-18 Because mycoplasmas are
More than 120 different species of Mycoplasma and 7 species of commonly isolated from the upper respiratory and genital tracts
Ureaplasma have been identified. The spectrum of mycoplasma of healthy dogs and cats, their role in ocular, upper respiratory
species that infect or colonize dogs and cats is incompletely tract, and urogenital tract disease has been difficult to deter-
understood because precise species identification has been dif- mine. Several studies have now identified an increased preva-
ficult (Box 40-1).3-7 Additional mycoplasma species are likely lence of mycoplasmas in cats with conjunctivitis or URTD when
to be discovered with the application of molecular methods in compared with healthy cats.13,19,20 Mycoplasmas can act as
the future. Some Mycoplasma spp. infect multiple host species. secondary invaders when other pathogenic viruses and bacteria
Mycoplasmas are often difficult to grow on cell-free media, and are present, or when normal host defenses are impaired by fac-
some, such as the hemotropic mycoplasmas, have never been tors such as underlying neoplasia or immunosuppressive drug

382

BOX 40-2
Diseases That May Be Associated with Mycoplasma
Infections in Dogs and Cats
Cats
Conjunctivitis
Upper respiratory tract disease
Pyothorax
Dogs
Urinary tract infections
Prostatitis
Epididymitis and orchitis
Both Cats and Dogs
Bronchopneumonia
Skin and soft tissue infections (abscesses)
Meningoencephalitis
Polyarthritis
therapy. When mycoplasmas are associated with systemic dis-
ease or pneumonia, young animals (less than 1 year of age) are
often involved. Some mycoplasma species appear to be more
pathogenic than others. For example, evidence has accumulated
that Mycoplasma cynos is associated with lower respiratory dis-
ease in dogs.21,22 In one study, M. cynos infection was associated
with increased severity of canine infectious respiratory disease,
younger age, and longer time spent in a kennel.22 Experimen-
tal infection with M. cynos leads to respiratory disease in dogs,
but infection with Mycoplasma gateae, Mycoplasma canis, and
Mycoplasma spumans does not.23 The role of mycoplasmas in
feline lower urinary tract disease and lymphoplasmacytic podo-
dermatitis has been investigated using molecular techniques, but
no association was found.24,25
Clinical Features
Signs and Their Pathogenesis
In order to colonize mammalian hosts, mycoplasmas must
adhere to host cells. Mycoplasma species that infect humans are
known to possess specific adhesins, but these have not been well
characterized in mycoplasmas from dogs and cats. Invasion of
deeper tissues and disease occurs as a result of immunosuppres-
sion or disruption of normal host barriers. Some mycoplasmas,
such as the human pathogen Mycoplasma pneumoniae, pro-
duce toxins, but whether similar virulence factors are present in
mycoplasmas isolated from dogs and cats is unknown. A num-
ber of pathogenic mycoplasma species of humans can invade
cells, which may contribute to organism persistence and dif-
ficulties associated with isolation in the laboratory. Transmis-
sion of pathogenic mycoplasmas such as M. cynos may occur in
overcrowded environments as a result of close contact or fomite
spread. The finding of identical strains in multiple dogs from a
kennel environment (using molecular typing methods) supports
the concept of transmission between dogs in this environment.26
The extent to which mycoplasmas survive in the environment
varies among mycoplasma species and has not been determined
for mycoplasmas that infect dogs and cats.
CHAPTER 40  Mycoplasma Infections 383
FIGURE 40-1  Ulcerative keratitis in a 4-year-old female spayed Persian. Involvement
of feline herpesvirus-1 was suspected, and Mycoplasma spp. was isolated from a corneal
swab. The significance of the Mycoplasma infection was unclear, but the ulcer healed after
topical treatment with topical ofloxacin and azithromycin drops. (Courtesy of the Univer-
sity of California, Davis, Veterinary Ophthalmology Service.)
Conjunctivitis and Upper Respiratory Tract Disease
Mycoplasma spp. are commonly detected in cats in associa-
tion with conjunctivitis and URTD. Concurrent infections with
other upper respiratory pathogens (such as Bordetella bron-
chiseptica, Chlamydia felis, and feline respiratory viruses) may
contribute to clinical signs. Stressors such as overcrowding and
unhygienic conditions may promote proliferation of mycoplas-
mas and their transmission between cats. Ulcerative keratitis has
been reported in association with Mycoplasma infection, but the
presence of corneal involvement may reflect the concurrent pres-
ence of feline herpesvirus 1 (FHV-1) or other factors that predis-
pose to secondary invasion by Mycoplasma spp. (Figure 40-1).
Pneumonia and Pyothorax
Virtually all dogs and cats harbor mycoplasmas in their upper
respiratory tracts. Fewer than 25% of healthy dogs harbor
mycoplasmas in their trachea or lungs. In one study myco-
plasmas were detected in tracheobronchial lavage specimens
from 21% of 28 cats with pulmonary disease, but not from
18 healthy cats.27,28 Mycoplasmas can be isolated from the
lower airways and lungs of dogs and cats with pneumonia,
sometimes in pure culture.29 Factors that predispose to the
development of mycoplasma pneumonia are not always appar-
ent.30 Clinical signs include variable fever, cough, tachypnea,
lethargy, and decreased appetite. Concurrent signs of URTD
have been reported in some affected cats.30 Rarely, mycoplas-
mas can be cultured from the pleural fluid of cats and dogs
with pyothorax, in pure culture or in mixed infections with
other organisms.31-33
Urogenital Tract
Mycoplasma and Ureaplasma spp. have been found in the semen
and urine of dogs with infertility and purulent epididymitis.34
The role of mycoplasmas in reproductive tract disease in dogs
and cats is unclear because they are commonly isolated from
the urogenital tract of healthy dogs and cats. Large numbers
of mycoplasmas (>105 CFU/mL) can occasionally be isolated
in pure culture from urine collected by cystocentesis from dogs
with prostatitis and lower urinary tract signs.14,34 Dogs with
mycoplasma UTIs often have other comorbidities that impair
384 SECTION 2  Bacterial Diseases

normal urinary defenses, such as urinary tract neoplasia, calculi,

or neurologic disease.14

Arthritis and Bacteremia

Isolated case reports exist of polyarthritis due to infection with
M. spumans or Mycoplasma edwardii (dogs) and M. gateae or
M. felis (cats). Synovial fluid analysis usually reveals the pres-
ence of large numbers of neutrophils. This may lead to diag-
nostic confusion with primary immune-mediated polyarthritis.
Systemic signs such as lethargy, fever, and inappetence usually
accompany lameness. Evidence of immune compromise, such
as underlying neoplasia or surgery, young age, or a history of
glucocorticoid treatment, has generally been present in dogs and
cats with mycoplasma polyarthritis. M. felis has also been iso-
lated from cats with monoarthritis, possibly secondary to bite
wound infections.17

Meningoencephalitis
Mycoplasmas may gain access to the central nervous system
after penetrating wounds, ascending infection from the middle
ear, or hematogenous spread. Meningoencephalitis associated
with M. felis infection was described in a 10-month-old cat that
showed signs of lethargy, fever, a head tilt, nystagmus, and tet-
raparesis.12 M. felis was isolated in pure culture from the CSF.
The cat also had evidence of otitis media-interna at necropsy,
but the role this played in development of the mycoplasma
infection was unclear. M. edwardii was isolated from the brain
of a 6-week-old dog with a sudden onset of seizures.8 Suppu-
rative and histiocytic meningitis was detected at necropsy, in
conjunction with evidence of a possible penetrating wound to
the skull. Recently, infection with Mycoplasma canis has been
associated with some cases of granulomatous meningoencepha-
lomyelitis and necrotizing meningoencephalitis in dogs,35 which
requires further study.
Physical Examination Findings
Physical examination findings in dogs and cats with mycoplasma
infections depend on the organ affected, as well as underlying
disease processes that facilitate opportunistic invasion by myco-
plasmas. Ocular infections in cats are characterized by serous
to mucopurulent ocular discharge, conjunctival hyperemia, and
possibly keratitis. Upper respiratory tract infections may result
in nasal discharge and sneezing, with or without conjunctivi-
tis. Fever, tachypnea, and increased lung sounds may be present
with pneumonia. Urinary tract infections may lead to clinical
signs of lower urinary tract disease, such as hematuria or stran-
guria. Mycoplasma arthritis may be manifested by fever, joint
pain and swelling, and sometimes local lymphadenopathy.9-11
Diagnosis
Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
Findings on the CBC, serum biochemistry panel, and urinaly-
sis in dogs or cats with Mycoplasma infections are nonspecific
and influenced by underlying immunocompromising disease
processes. Dogs and cats with pneumonia, mycoplasma bac-
teremia, and polyarthritis may have a neutrophilia with a left
shift and neutrophil toxicity, a degenerative left shift, mild
nonregenerative anemia, hypoalbuminemia, and evidence of
organ dysfunction such as increased liver enzyme activities and
azotemia.9,10,15
FIGURE 40-2  Ventrodorsal thoracic radiograph from a 3-month-old intact female
pug with Mycoplasma cynos pneumonia. There is consolidation of the lung lobes associ-
ated with the left hemithorax and the right middle lung lobe. A diffuse interstitial pattern
is present in the remainder of the right lung fields.
Diagnostic Imaging
Plain Radiography
Mycoplasma pneumonia may be characterized by interstitial
to alveolar patterns or lung lobe consolidation (Figure 40-2);
sometimes, mild pleural effusion is present. Radiographs of
the joints of dogs and cats with mycoplasma polyarthritis may
reveal periarticular soft tissue swelling. Erosive changes to the
subchondral bone have also been described.9
Microbiologic Tests
Assays available for diagnosis of mycoplasma infections in dogs
and cats are shown in Table 40-1.
Cytologic Examination
Mycoplasma spp. are often not visible by light microscopy
because of their small size. They do not stain with Gram stain
because they lack a cell wall. Cytologic examination of affected
body fluids (such as bronchoalveolar lavage fluid, synovial fluid,
and CSF) usually reveals a predominance of neutrophils with
fewer histiocytes and lymphocytes.9,10,12,29,30
Isolation and Identification
Specimens suitable for mycoplasma culture include synovial
fluid, blood, CSF, urine collected by cystocentesis, pleural fluid,
semen, and tracheobronchial or bronchoalveolar lavage speci-
mens. Swabs of the nasal cavity, conjunctiva, cornea, urethra,
vagina, or cervix can also be submitted, but interpretation of
results from these sites is difficult. If swabs are used, the swab
should be rubbed vigorously on the mucosa, because mycoplas-
mas are cell associated. Rapid transport to the laboratory is
 CHAPTER 40  Mycoplasma Infections 385

TABLE 40-1
Assays Available for Diagnosis of Mycoplasma Infections in Dogs and Cats
Assay Specimen Type Target Performance
Culture Swabs, body fluids, lavage Cultivable mycoplasma Some organisms grow on blood agar, but others require
specimens, tissue obtained species special mycoplasma media. The presence of unculti-
at necropsy vable mycoplasmas may lead to false-negative results.
Turnaround time may be slow (several days to weeks);
species differentiation and antimicrobial drug suscep-
tibility testing is often not performed. The significance
of positive results from the upper respiratory and
genital tract may be unclear.
PCR assays Swabs, body fluids, lavage Mycoplasma DNA Allows rapid detection of mycoplasmas. More sensitive
specimens, tissue obtained than culture for detection of fastidious or uncultivable
at necropsy mycoplasmas, although sensitivity and specificity may
vary between assays. Some assays are designed to only
detect certain pathogenic Mycoplasma species, such as
Mycoplasma cynos. Sequencing of the PCR product
may permit species identification. The clinical signifi-
cance of positive results may be unclear.

important, because mycoplasmas are susceptible to deteriora-

tion in the environment. The use of special transport media (e.g.,
Stuart’s medium or Amies medium without charcoal) for swab
or tissue specimens may increase yield of fastidious mycoplasma
species if a delay of several hours is likely between collection of
specimens and transport to the laboratory. Specimens should be
refrigerated if delayed transport is unavoidable. If the delay is
likely to be longer than 24 hours, specimens in transport media
should ideally be frozen at −80°C. Some mycoplasma species
will still grow in the laboratory if specimens are kept refriger-
ated for 2 to 3 days.
Some mycoplasmas grow on blood agar under routine aero-
bic or anaerobic conditions. Others require special mycoplasma
media, and some fail to grow under any laboratory conditions. FIGURE 40-3  Colonies of Mycoplasma bovirhinis showing the “fried egg” appear-
The rate of growth in culture varies from one Mycoplasma spe- ance that is typical of the growth of some Mycoplasma spp. in culture. (From The genera
cies to another; for some mycoplasmas, colonies are visible in Mycoplasma and Ureaplasma. In Songer JG Post KW, eds. Veterinary Microbiology Bacterial
2 or 3 days, whereas others require several weeks of incuba- and Fungal Agents of Animal Disease, 2 ed, St. Louis, MO: Saunders; 2005.)
tion before colonies appear. Examination of plates under the
light microscope may be required for detection of Mycoplasma
colonies, because the colonies range in size from 15 to 300 µm Antimicrobial susceptibility testing for mycoplasmas is labor
in diameter. The colonies of many mycoplasma species have a intensive and not routinely performed, and established break-
characteristic “fried egg” appearance (Figure 40-3). Biochemi- points are not available. When performed, the most widely used
cal properties can then be used to subgroup mycoplasmas, but method is microbroth dilution (see Chapter 3).36
species identification has traditionally required the use of spe-
cies-specific antisera. Because cross-reactions can occur between Molecular Diagnosis Using the Polymerase Chain Reaction
species even when antisera are used, molecular typing methods Some veterinary diagnostic laboratories offer PCR assays that
(such as 16S rRNA gene or 16S/23S rRNA intergenic spacer directly detect mycoplasma DNA in clinical specimens, without
region PCR assays and sequence analysis of the respective PCR the need for culture. Several conventional and real-time PCR
products) are now the method of choice for species identifica- assays have been used to detect mycoplasmas of dogs and
tion.3 Most veterinary diagnostic laboratories currently report cats.37-40 These assays may be specific for certain mycoplasma
only growth of a Mycoplasma species and do not identify species (such as M. cynos or M. felis) or they may be genus-
organisms to the species level unless specifically requested. It is specific assays that detect a variety of mycoplasma species.
difficult to interpret the significance of a positive mycoplasma PCR assays may be more sensitive than culture for detection of
culture from mucosal sites that can normally be colonized by fastidious mycoplasma species. Sequencing of the PCR prod-
mycoplasmas, including the lower respiratory tracts of dogs, so uct generated from genus-specific PCR assays may be used to
results must be interpreted in light of the clinical findings and identify the infecting species present, provided the PCR ampli-
the results of other diagnostic tests. con is sufficiently long. As with culture, the significance of a
386 SECTION 2  Bacterial Diseases
A
B weeks of treatment with pradofloxacin or doxycycline was
FIGURE 40-4  Histopathology of the inguinal subcutaneous tissues (A) and
sublumbar lymph node (B) from a 6-month-old, retrovirus–negative, male neu-
tered domestic shorthair with severe suppurative cellulitis, steatitis, dermatitis, and
myositis of the scrotal skin and subcutaneous tissue. A pure culture of a Mycoplasma
species was obtained from the lesion. The cat had been neutered 13 days previously
and was evaluated for a 2-day history of lethargy and reluctance to move. Physical
examination revealed fever (104.6°F [40.3°C]), conjunctivitis, generalized peripheral
lymphadenomegaly, and firm mass lesions in the inguinal area. In addition to suppu-
rative cellulitis and myositis, marked systemic lymph-node hyperplasia was present.
Hyperplasia was most profound in the sublumbar lymph nodes, which were 4 cm in
diameter.
positive PCR test result for Mycoplasma spp. may be unclear
given the role the frequent colonization of healthy animals
with mycoplasmas, so results must be interpreted with the
underlying disease process and the results of other diagnostic
tests.
Pathologic Findings
Gross pathology in dogs and cats with mycoplasmal diseases
usually reveals purulent material in affected tissues. Histo-
pathology shows predominantly neutrophilic inflammation,
although histiocytes may also be present. Reactive lymphade-
nopathy may also be evident as a result of immune stimulation
(Figure 40-4).
TABLE 40-2
Suggested Antimicrobial Drug Treatment for Mycoplasma
Infections in Dogs and Cats
Drug Dose (mg/kg) Route Interval (hours)
Doxycycline 5 to 10 PO or IV 12 to 24
Enrofloxacin* 5 PO or IV 24
Marbofloxacin 2.75 to 5.5 PO 24
Pradofloxacin† 5 to 10 (cats) PO 24
3 mg/kg (dogs)
*For cats, use enrofloxacin only when other alternatives are not possible
because of risk for irreversible blindness.
†Use of pradofloxacin is extra-label in dogs in the United States (see
Chapter 8). The dose for cats is for the oral suspension. The dose for the
tablet formulation is 3 mg/kg.
Treatment and Prognosis
Treatment of mycoplasma infections involves specific antimi-
crobial therapy (Table 40-2) as well as management of other
underlying disorders that predisposed to invasion by mycoplas-
mas. Tetracyclines or fluoroquinolones are active against most
mycoplasma isolates. The optimal duration is unknown, but
treatment for at least 2 weeks is reasonable. In a prospective
study of shelter cats with URTD and M. felis infection, a 14-day
course of doxycycline was more effective in causing reduction
in M. felis load as determined with real-time PCR than a 7-day
course, but there was no overall significant difference in the
reduction in clinical signs between the two groups.41 Neverthe-
less, 25% of the cats treated for 14 days were still infected after
that time. In another study of cats with conjunctivitis, three
required to eliminate Mycoplasma DNA.40 β-Lactam drugs are
not active against mycoplasmas, because they lack any pepti-
doglycan. Mycoplasmas are also intrinsically resistant to trime-
thoprim-sulfonamide combinations and rifampin. Some species
are resistant to macrolides.
Immunity and Vaccination
Mycoplasma spp. persist in tissues in the face of the immune
response. Mechanisms to evade the host immune response such
as antigenic variation of outer surface proteins have been identi-
fied in several mycoplasma species. Innate, humoral (especially
IgA), and cell-mediated immune responses may be required to
clear mycoplasma infections, with humoral mechanisms of criti-
cal importance. Vaccines to limit disease caused by pathogenic
mycoplasma species such as M. cynos are currently not avail-
able, although mycoplasma vaccines have been used with success
in food production animal species.
Prevention
Prevention of mycoplasma infections involves management or
reversal of disorders that predispose to opportunistic invasion
by mycoplasmas. Reduction of overcrowding and concurrent
infections in shelter environments may also reduce the inci-
dence and severity of mycoplasma infections (see Chapter 11).

Routine disinfectants are sufficient to inactivate mycoplasmas
that persist in the environment.
Public Health Aspects
The vast majority of mycoplasma species associated with dis-
ease in humans are different from those found in dogs and
cats. However, pharyngeal colonization with M. canis was
reported in a family and their pet dog.42 Mycoplasma felis
was isolated from the joints of a woman with common vari-
able immunodeficiency who developed septic arthritis.43 The
CASE EXAMPLE
Signalment: “Delilah”, a 3-month old intact female pug dog
from Reno, NV
History: Delilah was brought to the University of California,
Davis, Veterinary Medical Teaching Hospital for evaluation
of pneumonia. She had been purchased from a pet store 3
weeks before the date of evaluation. The owner noticed nasal
and ocular discharge when the dog was brought home from
the store, as well as a dry, nonproductive cough. Delilah was
taken to a local veterinary clinic where a diagnosis of “kennel
cough” was made. She was then treated with clavulanic acid–
amoxicillin (15 mg/kg PO q12h) for 2 weeks. However, the
cough persisted, and Delilah’s respiratory effort increased. She
was hospitalized at the local veterinary clinic and treated with
ticarcillin-clavulanate (50 mg/kg IV q8h), supplemental oxygen,
nebulization, and coupage. Radiographs showed infiltrates in
the left lung lobes and right middle lung lobe that worsened
over the course of hospitalization, so she was referred for
further evaluation and treatment. There had been no decrease
in appetite, vomiting, or diarrhea. She was fed a commercial
puppy food and had no history of ticks, travel, trauma, or toxin
exposure. The owner was not aware of previous vaccinations.
Physical Examination:
Body Weight: 2.2 kg
General: Quiet, alert, hydrated. T = 101.0°F (38.3°C), HR = 220
beats/min, RR = 60 breaths/min, slightly tacky mucous
membranes with a normal capillary refill time.
Eyes, Ears, Nose, and Throat: Mild serous oculonasal
discharge was present bilaterally. Conjunctival hyperemia
and chemosis were present, as well as scleral injection.
Musculoskeletal: BCS 4/9. Fully ambulatory with no
evidence of muscle atrophy. Poor body conformation was
present, characterized by medial deviation of the tarsi and
hyperextension of the carpi.
Respiratory: Tachypnea was present with marked abdominal
effort. Lung sounds were markedly increased, especially
on the left side. There were no crackles or wheezes.
A nonproductive cough occurred several times during the
examination.
All Other Systems: No clinically significant abnormalities were
detected.
Laboratory Findings:
Venous Blood Gas: pH = 7.348, pCO2 44 mm Hg, pO2 44.7 mm
Hg, HCO3 23.2 mmol/L, base excess −1.4 mmol/L, ionized
calcium 1.43 mmol/L, lactate 3.3 mmol/L.
CHAPTER 40  Mycoplasma Infections 387
woman had worked in an animal shelter, lived with a cat, and
had been bitten on the hand by a healthy cat 6 months before
she was evaluated. A mycoplasma was also isolated from a
cat scratch wound on the hand of a veterinarian.44 Although
Staphylococcus aureus was initially isolated from the wound,
it failed to resolve completely after treatment with erythro-
mycin. Subsequently, tenosynovitis developed, and a myco-
plasma was isolated. The identity of the mycoplasma could
not be determined using biochemical and serologic methods,
and whether a cat was truly the source of this infection was
not proven.
CBC:
HCT 35.4% (40%-55%)
MCV 68.6 fL (65-75 fL)
MCHC 31.1 g/dL (33-36 g/dL)
WBC 31,580 cells/µL (6000-13,000 cells/µL)
Neutrophils 6948 cells/µL (3000-10,500 cells/µL)
Band neutrophils 9790 cells/µL
Lymphocytes 9158 cells/µL (1000-4000 cells/µL)
Monocytes 4737 cells/µL (150-1200 cells/µL)
Eosinophils 947 cells/µL (150-1100 cells/µL)
Platelets 307,000 platelets/µL (150,000-400,000 platelets/µL).
Band neutrophils and mature neutrophils had evidence of
toxic changes.
Serum Chemistry Profile:
Sodium 147 mmol/L (145-154 mmol/L)
Potassium 4.9 mmol/L (3.6-5.3 mmol/L)
Chloride 104 mmol/L (108-118 mmol/L)
Bicarbonate 24 mmol/L (16-26 mmol/L)
Phosphorus 8.0 mg/dL (3.0-6.2 mg/dL)
Calcium 10.5 mg/dL (9.7-11.5 mg/dl)
BUN 10 mg/dL (5-21 mg/dL)
Creatinine 0.5 mg/dL (0.3-1.2 mg/dL)
Glucose 110 mg/dL (64-123 mg/dL)
Total protein 5.9 g/dL (5.4-7.6 g/dL)
Albumin 2.5 g/dL (3.0-4.4 g/dL)
Globulin 3.4 g/dL (1.8-3.9 g/dL)
ALT 12 U/L (19-67 U/L)
AST 38 U/L (19-42 U/L)
ALP 144 U/L (21-170 U/L)
Gamma GT 0 U/L (0-6 U/L)
Cholesterol 188 mg/dL (135-361 mg/dL)
Total bilirubin 0.0 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.045; pH 7.0, no protein, glucose, ketones,
bilirubin, WBC, or RBC were present. Rare triple phosphate
and a few sodium urate crystals were noted.
Imaging Findings:
Thoracic Radiographs: There was complete consolidation
of the lung lobes associated with the left hemithorax (see
Figure 40-2). Prominent air bronchogram formation was
noted in these lobes and in the right middle lung lobe, which
also appeared consolidated. Diffuse interstitial markings
were present in the remainder of the right lung fields.
Thoracic Ultrasound: Complete consolidation of the left lung
was the only abnormality detected.
Cytologic Findings: Transtracheal lavage: One direct smear
was evaluated that was highly cellular and contained a minimal
amount of mucus. Cells were essentially all neutrophils,
Continued
388 SECTION 2  Bacterial Diseases
which were primarily nondegenerate but occasionally mildly
to moderately degenerate. A single extracellular rod and
two neutrophils with intracellular structures that resembled
bacteria were found. Interpretation: Marked suppurative
inflammation.
Microbiologic Testing: Gram-stained direct smear of
transtracheal lavage fluid: Large numbers of neutrophils
were seen.
Aerobic and anaerobic bacterial culture and Mycoplasma cul-
ture (transtracheal wash fluid): Large numbers of Mycoplas-
ma spp.; DNA sequence analysis revealed 100% identity to
Mycoplasma cynos. No other aerobic or anaerobic organisms
were isolated.
Direct fluorescent antibody for distemper virus antigen (con-
junctival smear): Negative
Centrifugal zinc sulfate fecal flotation: Negative
Diagnosis: Mycoplasma cynos bronchopneumonia
Treatment: Before the culture results were available, Delilah
was treated with supplemental oxygen, nebulization,
coupage, and IV fluids. Treatment with ticarcillin-clavulanate
was continued for 3 days, then changed to enrofloxacin
(5 mg/kg PO q24h) and amoxicillin-clavulanate (19 mg/kg PO
q12h). Daily nebulization with gentamicin was also initiated,
because there had been no improvement and persistent
bordetellosis was considered possible. The following morning,
lung sounds were improved and Delilah was playful. Her nasal
discharge resolved and the frequency of cough reduced. The
CBC showed a regenerative anemia (hematocrit 33.2%, with
95,300 reticulocytes); 14,112 slightly toxic neutrophils/µL;
1008 band neutrophils/µL; 5796 lymphocytes/µL; and
3780 monocytes/µL. Growth of Mycoplasma spp. was
reported 5 days after specimens were submitted for culture.
Oxygen supplementation was withdrawn, and Delilah was
discharged on day 8 of hospitalization, although significant
radiographic improvement was not yet evident and an
occasional productive cough was still present. Treatment with
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Stenske KA, Bemis DA, Hill K, et al. Acute polyarthritis and septicemia
from Mycoplasma edwardii after surgical removal of bilateral adrenal
tumors in a dog. J Vet Intern Med. 2005;19:768-771.
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34. L’Abee-Lund TM, Heiene R, Friis NF, et  al. Mycoplasma canis
and urogenital disease in dogs in Norway. Vet Rec. 2003;153:
231-235.
35. Barber RM, Porter BF, Li Q, et  al. Broadly reactive polymerase
chain reaction for pathogen detection in canine granulomatous
meningoencephalomyelitis and necrotizing meningoencephalitis.
J Vet Intern Med. 2012;26:962-968.
36. Waites KB, Taylor-Robinson D. Mycoplasma and Ureaplasma.
In: Versalovic J, Carroll KC, Funke G, Jorgensen JH, Landry ML,
Warnock DW, eds. Manual of Clinical Microbiology. Washington,
DC: ASM Press; 2011:970-985.
37. Rebelo AR, Parker L, Cai HY. Use of high-resolution melting
curve analysis to identify Mycoplasma species commonly isolated
from ruminant, avian, and canine samples. J Vet Diagn Invest.
2011;23:932-936.
38. Soderlund R, Bolske G, Holst BS, et al. Development and evalu-
ation of a real-time polymerase chain reaction method for the
detection of Mycoplasma felis. J Vet Diagn Invest. 2011;23:
890-893.
39. Chalker VJ, Owen WM, Paterson CJ, et  al. Development of a
polymerase chain reaction for the detection of Mycoplasma felis in
domestic cats. Vet Microbiol. 2004;100:77-82.
40. Hartmann AD, Helps CR, Lappin MR, et  al. Efficacy of prado-
floxacin in cats with feline upper respiratory tract disease due to
Chlamydophila felis or Mycoplasma infections. J Vet Intern Med.
2008;22:44-52.
41. Kompare B, Litster AL, Leutenegger CM, et  al. Randomized
masked clinical trial to compare 7-day and 14-day course length of
doxycycline in the treatment of Mycoplasma felis infection in shel-
ter cats. Comp Immunol Microbiol Infect Dis. 2012:Dec 13 [Epub].
42. Armstrong D, Yu BH, Yagoda A, et al. Colonization of humans by
Mycoplasma canis. J Infect Dis. 1971;124:607-609.
43. Bonilla HF, Chenoweth CE, Tully JG, et al. Mycoplasma felis sep-
tic arthritis in a patient with hypogammaglobulinemia. Clin Infect Dis.
1997;24:222-225.
44. McCabe SJ, Murray JF, Ruhnke HL, et al. Mycoplasma infection of
the hand acquired from a cat. J Hand Surg Am. 1987;12:1085-1088.
CHAPTER 41

Hemoplasma Infections
Jane E. Sykes and Séverine Tasker

mycoplasmal pathogens Mycoplasma pneumoniae and Myco-

Overview of Hemoplasma Infections plasma genitalium.
First Described: Feline hemoplasmas, South Africa, 1942 At least three hemoplasma species infect domestic as well as
(Clark)1; canine hemoplasmas, Germany, 1928 (Kikuth)2 wild cats, Mycoplasma haemofelis, ‘Candidatus Mycoplasma
haemominutum,’ and ‘Candidatus Mycoplasma turicensis.’ The
Cause: Mycoplasma haemofelis, ‘Candidatus Mycoplasma
‘Candidatus’ prefix is applied to newly discovered hemoplasmas
haemominutum,’ ‘Candidatus Mycoplasma turicensis’
until more information is available to support their classifica-
(cats). The most prevalent hemoplasmas in dogs are
tion. This is because hemoplasmas cannot be cultured in the
Mycoplasma haemocanis and ‘Candidatus Mycoplasma
laboratory, which limits full characterization of these organ-
haematoparvum.’
isms. M. haemofelis (previously the Ohio strain, or large form
Affected Hosts: Cats and wild felids, dogs and wild canids; of Haemobartonella felis) is the most pathogenic organism and
other hemoplasma species cause disease in a variety of can cause moderate to severe hemolytic anemia in immunocom-
other animal species. petent cats. The resulting disease has been referred to as feline
Geographic Distribution: Worldwide infectious anemia. Using cytologic evaluation of blood smears,
M. haemofelis organisms are cocci that sometimes form short
Mode of Transmission: Experimentally, transmission can
chains of three to six organisms (Figure 41-1). M. haemofelis is
occur through ingestion or injection of infected blood;
the least prevalent of the three feline hemoplasmas. It has been
the mode of transmission in the field uncertain. Biting or
found using PCR in 0.5% to 5% of sick cats at veterinary hos-
aggressive interactions are a suspected mode, and pos-
pitals. The whole genome sequences of M. haemofelis and M.
sibly vertical transmission. Ticks are suspected to transmit
haemominutum have been determined.3-5
Mycoplasma haemocanis. Other unidentified arthropod
‘Ca. M. haemominutum’ (previously the California strain or
vectors may also be involved.
small form of H. felis) is generally smaller than M. haemofelis
Major Clinical Signs: Fever, lethargy, inappetence, weakness, and has not clearly been associated with disease in immunocom-
pallor. petent cats. Using cytologic evaluation of blood smears, ‘Ca.
Differential Diagnoses: Cytauxzoonosis, FIV infection, FeLV M. haemominutum’ are small cocci, 0.3 to 0.6 µm in diameter,
infection, feline infectious peritonitis, primary immune- although M. haemofelis and ‘Ca. M. haemominutum’ cannot
mediated hemolytic anemia, Heinz body hemolytic ane-
mia, inherited erythrocyte disorders such as pyruvate
kinase deficiency and the red cell fragility disorder of
Abyssinian and Somali cats
Human Health Significance: DNA sequences that match
those found in Mycoplasma haemofelis were detected in a
human co-infected with HIV and Bartonella henselae. DNA
sequences of other animal hemoplasmas have also been
detected in humans.

Etiology and Epidemiology

Hemotropic mycoplasmas (hemoplasmas) are small (0.3-0.8
µm), unculturable mycoplasmas that reside on the surface of
erythrocytes and can cause variable degrees of hemolytic anemia
in infected hosts. Hemoplasmas infect a wide variety of mam-
malian species, including humans, and have a worldwide distri-
bution. Although previously classified as Haemobartonella and
Eperythrozoon spp., sequence analysis of the 16S rRNA genes FIGURE 41-1  Romanowsky-stained blood smear from an 8-year-old male neutered
of these organisms has shown that they are closely related to the domestic shorthair cat showing epierythrocytic bacteria typical of Mycoplasma haemofelis
pneumoniae group of mycoplasmas, which includes the human (arrows).

390

always reliably be distinguished by this method. ‘Ca. M. haemo-
minutum’ is common in the cat population worldwide, infect-
ing as many as 20% of cats that visit veterinary hospitals.6-8
Infection of cats with ‘Ca. M. haemominutum’ results in a mild
decrease in hematocrit. There is some evidence that ‘Ca. M. hae-
mominutum’ may play a role in disease. For example, cats that
are co-infected with FeLV and ‘Ca. M. haemominutum’ develop
more significant anemia than cats infected with ‘Ca. M. hae-
mominutum’ alone, and progression to FeLV-induced myelo-
proliferative disease occurred more rapidly.9 There are also
descriptions of cats with hemolytic anemia in which the only
recognized cause of anemia was ‘Ca. M. haemominutum.’10 ‘Ca.
M. haemominutum’ is commonly found in co-infections with
‘Ca. M. turicensis’ or M. haemofelis. Mixed infections with all
three feline hemoplasma species also have been described.
‘Ca. M. turicensis’ was first described in a cat from Switzer-
land that had severe intravascular hemolysis (turicensis pertains
to Turicum, the Latin name of Zurich).11 Infections with ‘Ca. M.
turicensis’ have since been detected worldwide.8,12-14 ‘Ca. M. turi-
censis’ has never been seen using light microscopic examination
of blood smears, and organism loads in infected cats are typically
low. Infection with ‘Ca. M. turicensis’ is slightly more prevalent in
the cat population than infection with M. haemofelis. Most stud-
ies show a prevalence of 0.5% to 10% in sick cats that visit vet-
erinary hospitals. The pathogenic potential of this organism is not
fully understood. Inoculation of an immunosuppressed cat with
‘Ca. M. turicensis’ resulted in severe anemia,11 but little or no ane-
mia occurs in immunocompetent cats after inoculation with ‘Ca.
M. turicensis.’ Cofactors, such as co-infection with other hemo-
plasmas or concurrent immunosuppression, may influence the
development of anemia in cats infected with ‘Ca. M. turicensis.’
Infection of cats by hemoplasmas is strongly associ-
ated with male sex, nonpedigree status, and outdoor access
(Box 41-1).8,12,15,16 Infection with ‘Ca. M. haemominutum’ is
more prevalent in older cats, presumably because the chance
BOX 41-1
Risk Factors Associated with Infection by Feline Hemotropic
Mycoplasmas as Determined Using Species-Specific
Real-Time PCR Assays
Mycoplasma haemofelis
Younger age
Male sex
FIV seroreactivity
FeLV seroreactivity
‘Candidatus Mycoplasma haemominutum’
Older age
Male sex
FIV seroreactivity
Nonpedigree status
Outdoor access
‘Candidatus Mycoplasma turicensis’
Male sex
From Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal
Medicine, 7 ed, St. Louis, MO: Saunders; 2011.
CHAPTER 41  Hemoplasma Infections 391
of acquiring persistent subclinical infection increases over time.
In contrast, young cats may be more likely to develop disease
after infection by M. haemofelis. Some studies, but not others,
have shown an association between retrovirus and hemoplasma
infections. Cats infected with M. haemofelis in the United States
were 6 times more likely to be FIV infected than cats negative
for hemoplasmas.15
Several hemoplasma species also infect dogs. Infection with
Mycoplasma haemocanis (previously Haemobartonella canis) has
been associated with hemolytic anemia in splenectomized dogs,
and rarely in dogs with other immunosuppressive disease or con-
current infections. The 16S rRNA gene of M. haemocanis has the
same sequence as M. haemofelis, but the whole genome sequence
of M. haemocanis distinguishes it as a different species.17 M. hae-
mocanis is a coccoid organism that often forms long chains of
organisms (Figure 41-2). The prevalence of this infection is par-
ticularly high in kennel-raised dogs, which are often infected sub-
clinically.18 In the southwestern United States, infection was also
prevalent among coyotes.19
Three additional hemoplasma species have been detected in
dogs. ‘Candidatus Mycoplasma haematoparvum’ is a small (0.3
µm) coccoid organism that resembles ‘Ca. Mycoplasma haemo-
minutum’ morphologically as well as genetically (Figure 41-3).
‘Ca. M. haemominutum’ has also been detected in several dogs
using PCR assays, and organisms that resemble ‘Ca. M. haema-
toparvum’ and ‘Ca. M. haemominutum’ have been detected in
European wolves and bush dogs from Brazil.20 The ovine hemo-
plasma Mycoplasma ovis was detected in the spleens of a small
number of dogs from the southeastern United States, and the
bovine hemoplasma ‘Ca. Mycoplasma haemobos’ was detected
in a dog from northern Australia.21,22 The clinical importance of
these hemoplasma species in dogs remains unclear.
The mode of transmission of hemoplasmas remains unclear.
To some extent, fleas and other arthropod vectors may be capa-
ble of transmitting feline hemoplasmas,23 but experimental evi-
dence for vector-borne transmission of feline hemoplasmas is
weak. Transmission of M. haemocanis by the brown dog tick,
Rhipicephalus sanguineus, has been demonstrated experimen-
tally, although this was before PCR assays were available to
confirm infection.24 Geographic variation in the prevalence of
FIGURE 41-2  Romanowsky-stained blood smear showing chains of epierythrocytic
bacteria typical of Mycoplasma haemocanis. A Howell-Jolly body is also present (arrow).
392 SECTION 2  Bacterial Diseases
FIGURE 41-3  Blood smear from a dog showing ‘Candidatus Mycoplasma haema-
toparvum’ (small arrows) and a Howell-Jolly Body (large arrow).
hemoplasma infection in dogs and cats supports a role for arthro-
pod vectors in transmission. For example, in Europe, infection
with M. haemocanis is more prevalent in Mediterranean coun-
tries, which follows the distribution of Rh. sanguineus. Vertical
(e.g., transplacental) spread has also been hypothesized and has
been documented for bovine hemoplasmas. Biting has been sug-
gested as a means of transmission of feline hemoplasmas, and
the strong male sex predilection, recent history of cat bite absces-
sation in some cats, and association with FIV infection in some
studies supports this mode. Additionally, studies in Switzerland
found that subcutaneous inoculation of blood that contained
‘Ca. M. turicensis’ resulted in transmission, whereas inoculation
of saliva that contained ‘Ca. M. turicensis’ did not.25 This sug-
gests that hemoplasma transmission by social contact (saliva via
mutual grooming etc.) is less likely than transmission by aggres-
sive interaction (blood transmission during a cat bite incident).
Because infection can also be transmitted by ingestion of blood,
it may be that the biting cat (rather than the bitten cat) is most at
risk for acquisition of infection. Transmission of M. haemocanis
through ingestion of infected blood has also been described,26 so
aggressive interactions between dogs may also have the poten-
tial to transmit hemoplasmas; however, this mode of transmis-
sion remains to be proven in field circumstances. Transmission
can also occur following blood transfusion.
Clinical Features
Signs and Their Pathogenesis
Because only M. haemofelis infection is associated with signifi-
cant anemia in cats, only the pathogenesis of disease induced by
this species is described here. After experimental infection, there
is a delay of 2 to 34 days before the onset of clinical signs. Anemia
then occurs and persists for about 18 to 30 days (acute phase),
although its severity and chronicity vary considerably between
infected cats. Anemia predominantly results from extravascu-
lar hemolysis, and the onset of anemia is usually followed by
a strong regenerative response with reticulocytosis. The organ-
ism resides in an indentation on the erythrocyte surface (Figure
41-4). Production of both cold and warm reactive erythrocyte-
bound antibodies, increased osmotic fragility, and decreased
FIGURE 41-4  Electron micrographs showing epierythrocytic location of Mycoplasma
haemofelis.
erythrocyte life span have been noted in infected cats.27-29 The
organisms can also “bridge” adjacent erythrocytes, which might
promote splenic trapping and removal of red blood cells. In
contrast, erythrocyte-bound antibody formation and significant
reticulocytosis have not been documented in cats infected with
‘Ca. M. haemominutum’ or ‘Ca. M. turicensis.’
Anemia can result in signs of lethargy, inappetence, pallor,
and weakness. Some owners report that their cats eat dirt or
litter or lick cement (pica). Weight loss and dehydration can
occur. Rapid development of anemia may result in neurologic
signs, vocalization, collapse, and death. In some infected cats,
cyclical changes in the hematocrit and numbers of infected
erythrocytes occur, with sharp declines in the hematocrit corre-
lating with appearance of large numbers of organisms in blood
smears.30-32 The proportion of erythrocytes associated with vis-
ible organisms on blood smears can decline from 90% to less
than 1% in less than 3 hours.33,34 These dramatic fluctuations
in organism numbers appear to result from rapid replication of
M. haemofelis, followed by rapid clearance from the blood as a
result of the host’s immune response. Repeated antigenic varia-
tion in M. haemofelis may allow the organism to again prolifer-
ate in the face of this immune response, which contributes to
ongoing fluctuations in organism numbers.
In cats that survive the acute phase, the hematocrit then
returns to normal or near-normal (recovery phase), and organ-
isms can no longer be visualized in blood smears. It is possible
that at least some of these recovered cats remain persistently
infected, whereby the organism evades the host immune system,
and that recrudescence of anemia may follow stress, pregnancy,
intercurrent infection, or neoplasia.32,33 However, evidence is
 CHAPTER 41  Hemoplasma Infections 393

accumulating that differences may exist in the ability of hemo-

plasmas to persist within the host, the carrier state being more
frequent for ‘Ca. M. haemominutum’ but less frequent for M.
haemofelis and ‘Ca. M. turicensis.’ In cats, splenectomy has a
variable effect on the course of hemoplasmosis. Recrudescence
of anemia and bacteremia has been documented in some chroni-
cally infected cats after splenectomy, although other studies sug-
gest that splenectomy increases the number of visible organisms
in blood smears without causing significant anemia.32,34 Infec-
tion of splenectomized cats with ‘Ca. M. haemominutum’ does
not appear to enhance the pathogenicity of this organism.35
Dogs that develop clinical hemoplasmosis have usually had a
history of splenectomy or, less commonly, concurrent immuno-
suppressive illness or drug treatment or co-infections with other
bloodborne pathogens such as Babesia spp. and Ehrlichia canis.
Clinical signs include weakness, lethargy, and pallor, and some
dogs are inappetent.
FIGURE 41-5  Mild icterus of the nictating membrane in an 8-year-old male neutered
Physical Examination Findings domestic shorthair with hemolytic anemia secondary to Mycoplasma haemofelis infection.
Physical examination findings in cats with acute M. haemofelis
infection include fever, weakness, mucosal pallor, tachypnea,
tachycardia, and weak or bounding femoral pulse quality. Other
physical examination abnormalities can include dehydration,
cardiac murmurs, splenomegaly, and occasionally mild icterus
(Figure 41-5). Moribund cats may be hypothermic.
Dogs with hemoplasmosis can show lethargy and mucosal
pallor. Fever is usually absent.
Cats infected with ‘Ca. M. haemominutum’ or ‘Ca. M. turi-
censis,’ and dogs infected with ‘Ca. M. haematoparvum’ gener-
ally appear healthy unless concurrent disease is present.

Diagnosis
Laboratory Abnormalities
Complete Blood Count
The most characteristic CBC abnormality in dogs and cats
infected with M. haemocanis and M. haemofelis, respectively, is
regenerative anemia, with macrocytosis, anisocytosis, reticulo-
cytosis, polychromasia, Howell-Jolly bodies, and, especially in
cats, sometimes marked normoblastemia. Manual reticulocyte
counts should be interpreted with caution in cats infected with
M. haemofelis, because both hemoplasma-infected erythrocytes
have the same appearance as reticulocytes in blood smears stained
with methylene blue. Autoagglutination may be noted in blood
smears. Nonregenerative anemia can be present when sufficient FIGURE 41-6  Ventrodorsal abdominal radiograph from an 8-year-old male neutered
time for a regenerative response has not yet elapsed, or as a result domestic shorthair cat with hemolytic anemia secondary to Mycoplasma haemofelis Infec-
of concurrent FeLV infection or any other disease that suppresses tion. There is marked splenomegaly (arrows).
the regenerative response. Concurrent occult hemoplasma infec-
tion should be considered in any anemic FeLV-positive cat, even
in the absence of reticulocytosis. Neutrophil counts may be nor- Diagnostic Imaging
mal, elevated, or low, and lymphopenia may be present. Throm- The most common radiographic finding in cats with hemoplas-
bocytopenia can also occur in affected dogs and cats, but often mosis is splenomegaly (Figure 41-6). The spleen may be absent
the platelet count is within the reference range. in affected dogs because of previous splenectomy.

Serum Biochemical Tests Microbiologic Tests

The serum chemistry profile in cats with hemoplasmosis may
reveal increased activities of ALT and AST as a result of hypoxia;
metabolic acidosis; mild to moderate hyperbilirubinemia; and
prerenal azotemia. Hyperproteinemia may be seen in some cats.
Urinalysis
The urinalysis is usually unremarkable; bilirubinuria may be
present in hyperbilirubinemic cats.
Assays available for diagnosis of hemoplasmosis in dogs and
cats are listed in Table 41-1.
Cytologic Examination of Blood Smears
Cytologic detection of hemoplasmas has low sensitivity and
specificity, especially for diagnosis of feline infectious anemia.
M. haemofelis is visible less than 50% of the time in acutely
infected cats, because organisms can disappear for days before
394 SECTION 2  Bacterial Diseases
TABLE 41-1
Assays Available for Diagnosis of Hemoplasmosis in Dogs and Cats
Assay Specimen Type Target
Cytologic Blood smears Hemoplasmas
examination
PCR Whole blood, blood Hemoplasma DNA
smears
reappearing on blood smears over the course of infection.
Because organisms detach from erythrocytes within hours
of blood collection, fresh blood smears should be submitted.
False-positive diagnoses occur when stain artifacts such as pre-
cipitate are confused with organisms; thus, careful staining with
a properly prepared, uncontaminated Romanowsky-type stain
solution is essential. Organisms also need to be distinguished
from basophilic stippling and Howell-Jolly bodies. ‘Ca. M. hae-
mominutum’ is small and is not usually visible in chronically
infected cats. ‘Ca. M. turicensis’ has never been seen on blood
smears with light microscopy. Hemoplasmas are visible in many
clinically affected dogs, and because M. haemocanis tends to
form chains, it is more readily differentiated from stain artifacts
and erythrocyte morphologic changes than is M. haemofelis.
However, hemoplasmas are not usually visible in chronically
infected dogs or in dogs infected with ‘Ca. M. haematoparvum.’
Culture
Attempts to isolate and grow feline hemotropic mycoplasmas in
the laboratory have been unsuccessful, so blood culture cannot
be used for diagnosis of hemoplasmosis.
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays are currently the tests of choice for diagnosis of
hemoplasmosis in dogs and cats. Numerous assays have been
described to date, which are generally based on detection of
the 16S rRNA gene. These are significantly more sensitive than
blood smear evaluation, although they occasionally do not
detect infection in apparently healthy carrier cats when organism
numbers in the blood are below the detection limit of the PCR
assay.6,30,36,37 Both conventional and real-time PCR assays have
been described. Some conventional PCR assays do not differ-
entiate between M. haemofelis and ‘Ca. M. turicensis,’ because
the primers used in the assay generate the same-sized PCR prod-
uct when either or both of these species are present. Real-time
PCR assays are usually species specific. Because the pathogenic
potential of each hemoplasma species differs, the laboratory
should be consulted to determine the species specificity of the
assay(s) offered. Alternative causes of anemia should always be
considered, especially in cats that test positive for ‘Ca. M. hae-
mominutum’ or ‘Ca. M. turicensis,’ because infection with these
organisms is not commonly associated with anemia. Indeed,
all PCR results should be interpreted in conjunction with the
patient’s clinical signs, the degree and nature of the anemia pres-
ent, and any concurrent signs or diseases present that could be
contributing to the clinical signs. Dried blood smears can also
Performance
Poor sensitivity, especially in affected cats. Organisms are seen on
blood smears in fewer than 50% of sick cats. False positives can
occur when stain precipitate, drying artifact, basophilic stippling,
and Howell-Jolly bodies are misinterpreted as hemoplasmas.
Sensitivity and specificity can vary depending on assay design.
Results must be interpreted in light of the species detected and
the clinical findings. Mycoplasma haemofelis and Mycoplasma
haemocanis are most likely to be associated with anemia.
be tested but are less sensitive than liquid whole blood speci-
mens.38 PCR assays designed to detect M. haemofelis generally
also detect M. haemocanis because of the high 16S rRNA gene
sequence homology between these species, but a separate assay
is required for detection of ‘Ca. M. haematoparvum.’
Serologic Diagnosis
The development of serologic tests for diagnosis of hemoplas-
mosis has been hampered by the inability to culture hemoplas-
mas in the laboratory. However, the application of molecular
techniques has led to the identification of antigens for serologic
assay development. Specifically, an ELISA that detects antibod-
ies to an immunodominant protein of M. haemofelis, DnaK,
has been developed. Cats produce antibodies to this antigen
after infection,39,40 and in a study that examined the immune
response to ‘Ca. M. turicensis’ infection, antibody titers to
DnaK declined with antimicrobial drug treatment.39 Serologic
cross-reactivity occurs to this antigen for all three feline hemo-
plasma species40; therefore, the assay does not discriminate
between infection with M. haemofelis and other hemoplasmas,
but determination of acute and convalescent titers may be help-
ful to distinguish acute from chronic infection. Currently this
assay is available only on a research basis.
Pathologic Findings
Generalized pallor, splenomegaly, and, in some cats, icterus are
the main gross necropsy finding in cats that die or are eutha-
nized as a result of hemoplasmosis. Histopathologic findings
include extramedullary hematopoiesis, follicular hyperplasia,
and erythrophagocytosis within the spleen.
Treatment and Prognosis
Treatment is indicated for cats and dogs with clinical signs
and laboratory abnormalities consistent with hemoplasmosis.
Although controversial, treatment could also be considered
for apparently healthy cats that test positive for M. haemofelis
in view of the potential for recrudescence of disease with this
hemoplasma species. It may be difficult or impossible to eradi-
cate infection with some hemoplasma species or strains, espe-
cially ‘Ca. M. haemominutum.’ Indeed, no antibiotic treatment
regime consistently eliminates hemoplasma infection in cats.
The treatment of choice for hemoplasmosis is doxycycline
for a minimum of 2 weeks (see Chapter 8 for information on
doxycycline) (Table 41-2); some have suggested using longer
courses of treatment (6 to 8 weeks) to ensure that infection is

TABLE 41-2
Suggested Drug Dosages for Treatment of Hemoplasmosis
in Cats
Dose Interval
Drug (mg/kg) Route (hours) Duration
Doxycycline 10 PO 24 2 weeks
Marbofloxacin 2.75 to 5.5 PO 24
Pradofloxacin* 5 to 10 PO 24
Enrofloxacin† 5 PO 24
*Dose listed is for the oral suspension for cats.
†Only when other alternatives are not available because of potential for
irreversible blindness when enrofloxacin is used in cats.
eliminated. Real-time PCR could be used to monitor response
to antibiotic treatment if quantitative information is avail-
able. Fluoroquinolones are an alternative; marbofloxacin and
pradofloxacin,41 when available, are both efficacious. Enro-
floxacin is also usually effective,42 but diffuse retinal degenera-
tion and acute blindness have been reported following the use
of this fluoroquinolone and, although this is rare, caution must
be exercised with its use in cats. The response of the different
hemoplasma species, and indeed different strains of the same
species, to antibiotics varies. Most successful studies that have
demonstrated treatment efficacy have evaluated the response
of M. haemofelis to treatment. Affected dogs and cats may
also require supportive treatment with IV crystalloids or blood
products. Clinical improvement usually occurs within 2 to
3 days. The use of immunosuppressive doses of glucocorticoids
to suppress the associated immune-mediated hemolytic process is
controversial, given that glucocorticoids can cause reactivation of
latent infection, but may be necessary in cats that fail to respond
to antimicrobial therapy alone, or when the diagnosis is uncer-
tain, especially if erythrocyte-bound antibodies are identified.
Doxycycline is the drug of choice for treatment of M. hae-
mocanis infection. Treatment of a splenectomized dog with
oxytetracycline for approximately 1 month was associated
with clinical improvement, but infection persisted as deter-
mined with quantitative PCR, and relapse occurred when
treatment was withdrawn.43 Infection also persisted after
treatment with enrofloxacin, despite eventual recovery from
anemia. In contrast, in M. haemocanis–infected dogs with
intact spleens, doxycycline treatment for 4 weeks appeared to
result in resolution of infection as determined by PCR assay,
whereas ‘Ca. M. haematoparvum’–infected dogs remained
PCR positive.19 A splenectomized dog was apparently cured
of M. haemocanis infection after 12 weeks of doxycycline
treatment.44
CHAPTER 41  Hemoplasma Infections 395
Immunity and Vaccination
Little information is available in regard to the immune response
to hemoplasma infection, and no vaccines are available. Because
co-infections with multiple hemoplasma species occur, infection
with one species does not appear to protect against infection
with other species.
Prevention
Inadvertent transmission of hemoplasmas by transfusion of
blood from carrier cats has been documented, and so all blood
donor dogs and cats should be tested for hemoplasmas with
PCR assays. In the future, screening with serologic assays may
also be used to identify potential donor animals. Whenever pos-
sible, cats and dogs that are PCR negative for any of the known
hemoplasma species should be excluded as donors. Given the
high prevalence of subclinical ‘Ca. M. haemominutum’ infec-
tion among cats, in some circumstances it may not be possible
to find a donor that is ‘Ca. M. haemominutum’ negative. At this
time, it is unclear whether transfusion of blood from cats that are
infected with ‘Ca. M. haemominutum’ is associated with adverse
outcome, but it remains a possibility. Keeping cats indoors is
also likely to prevent infection, as outdoor access has been iden-
tified as a risk factor. Control of fleas and ticks is recommended.
Public Health Aspects
Organisms that resembled hemoplasmas were identified in
human blood smears decades ago. Molecular techniques that
were originally designed for detection of canine and feline hemo-
plasmas have since been applied to human blood, and a variety
of hemoplasma species have now been identified. In addition,
application of these methods to archived human blood smears
believed to contain hemoplasmas based on cytological evaluation
yielded negative results, suggesting the possibility of historical
misdiagnosis.45 Infection with a novel hemoplasma, designated
‘Ca. Mycoplasma haemohominis,’ has been identified in a per-
son with hemolytic anemia.46 M. haemofelis DNA was identi-
fied in a person from Brazil who was co-infected with HIV and
Bartonella henselae.47 In addition, DNA of Mycoplasma ovis
was detected in a Texas veterinarian who was co-infected with
B. henselae.48 Mycoplasma suis DNA has been found in farm
workers from China.49 The identification of the DNA of animal
hemoplasmas in humans should be viewed cautiously because
only a small portion or portions of the hemoplasma genome
was detected in each of these case reports. M. haemocanis and
M. haemofelis have identical 16S rRNA gene sequences, but
different host tropisms, and so the organisms detected in these
humans may not necessarily be the same as those that infect
domestic animal species. Until further information is available,
veterinarians should handle blood from animals with caution.
396 SECTION 2  Bacterial Diseases
CASE EXAMPLE
Signalment: “Shadow,” an 8-year-old male neutered
domestic shorthair from Fairfield, CA
History: Shadow was brought to the University of California,
Davis, Veterinary Medical Teaching Hospital emergency
service for treatment of severe anemia. The owners reported
that he had disappeared 5 days previously. The evening that
he was brought to the emergency service, he was found
splayed across a fence and was unwilling to move, and
vocalizing and salivating profusely. He was immediately
taken to a local emergency clinic where he was found to
be obtunded, pale, and severely dehydrated. His pulse was
180 beats/min, respiratory rate was 60 breaths/min, and
temperature was 94.0°F (34.4°C). A systolic blood pressure
was 80 mm Hg. A PCV was 12%. Shadow’s condition was
stabilized after treatment with supplemental oxygen, active
warming, and IV crystalloid fluids (Normosol-R, 50 mL over
30 minutes, then 25 mL/hr). He tested negative for FIV
antibody and FeLV antigen with an in-practice ELISA assay.
Shadow was an indoor-outdoor cat that was acquired as a
stray kitten from the owner’s backyard. There had been no
previous history of illness.
Physical Examination:
Body Weight: 6 kg
General: Obtunded, laterally recumbent. T = 97.8°F (36.6°C), HR
= 150 beats/min, RR = 60 breaths/minute, purring, pale and
tacky mucous membranes with no detectable capillary refill,
weak pulses.
Integument: No ectoparasites were noted. The pinnae and
ventral abdominal skin were icteric.
Eyes, Ears, Nose, and Throat: Moderate dental calculus was
present. Pallor and mild icterus of the mucous membranes,
nictitans, and sclera was noted (see Figure 41-5).
Musculoskeletal: Body condition score was 7/9.
Cardiovascular: Weak pulses. No other clinically significant
abnormalities were detected. No murmurs were detected
but the heart sounds were difficult to auscultate because of
persistent purring.
Respiratory: Tachypneic; auscultation was limited because of
purring.
Gastrointestinal and Genitourinary: Nonpainful abdomen.
Moderate cranial organomegaly. Urinary bladder not
palpable.
Neurologic: A full neurologic examination was not performed
but no abnormalities were detected on examination of
cranial nerve functions.
Lymph Nodes: No clinically significant abnormalities
detected.
Laboratory Findings:
PCV/TPP: 14%/7.3 g/dL, icteric plasma.
Saline Slide Agglutination Test: Negative for
autoagglutination.
Blood Smear Examination: Marked polychromasia,
normoblastosis, and mild anisocytosis were present.
Coccoid bodies that resembled hemoplasmas were present
in association with erythrocytes (see Figure 41-1).
Electrolyte/Acid-Base Panel (Venous Blood): HCO3 = 14
mmol/L, pH = 7.365, pCO2 = 21.9 mm Hg, pO2 = 35.4 mm Hg,
Hb = 2.8 g/dL, O2 = 59.5%, potassium = 4.0 mmol/L, sodium =
144 mmol/L, ionized calcium = 1.2 mmol/L, glucose = 157 mg/
dL, lactate = 8.4 mmol/L, base excess = −12.2 mmol/L.
Imaging Findings:
Thoracic Radiographs: No clinically significant abnormalities
were detected.
Abdominal Radiographs: The spleen was markedly enlarged
(See Figure 41-6). The liver was mildly enlarged. Small
mineral densities were present within ingesta and feces in
the stomach and colon, respectively.
Treatment: The cat was placed on a heating pad and
transfused with 1 unit of packed RBC, and supplemental
oxygen was administered, after which heart rate increased
to 200 beats/min and respiratory rate decreased to 40
breaths/min. Rectal temperature increased to 103.7°F
(39.8°C) during the transfusion, and so dexamethasone
was administered because of the concern for a transfusion
reaction (0.2 mg/kg IV once). The cat was also treated with
enrofloxacin (5 mg/kg IV q24h) (see comments section later)
for suspected hemoplasmosis. The results of laboratory
testing after transfer to the internal medicine service the day
after admission are shown here.
CBC (Post Transfusion and Antimicrobial Treatment):
HCT 12.7% (30%-50%)
MCV 76.5 fL (42-53 fL)
MCHC 26.8 g/dL (30-33.5 g/dL)
Reticulocytes 155,600 cells/µL (7000-60,000 cells/µL)
Nucleated RBC 39/100 WBC (0/100 WBC)
Corrected WBC 9800 cells/µL (4500-14,000 cells/µL)
Neutrophils 8134 cells/µL (2000-9000 cells/µL)
Band neutrophils 98 cells/µL
Lymphocytes 784 cells/µL (1000-7000 cells/µL)
Monocytes 784 cells/µL (50-600 cells/µL)
Eosinophils 0 cells/µL (150-1100 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL)
Platelets 142,000/µL (180,000-500,000 platelets/µL).
Cytologic examination revealed marked anisocytosis, marked
polychromasia, few Howell-Jolly bodies, and many mac-
rocytes and microcytes. No hemoplasmas were identified.
Serum Chemistry Profile:
Sodium 143 mmol/L (151-158 mmol/L)
Potassium 5.2 mmol/L (3.6-4.9 mmol/L)
Chloride 107 mmol/L (117-126 mmol/L)
Bicarbonate 11 mmol/L (15-21 mmol/L)
Phosphorus 6.6 mg/dL (3.2-6.3 mg/dL)
Calcium 9.6 mg/dL (9.0-10.9 mg/dL)
BUN 47 mg/dL (18-33 mg/dL)
Creatinine 1.2 mg/dL (1.1-2.2 mg/dL)
Glucose 98 mg/dL (63-118 mg/dL)
Total protein 7.2 g/dL (6.6-8.4 g/dL)
Albumin 3.0 g/dL (2.2-4.6 g/dL)
Globulin 4.2 g/dL (2.8-5.4 g/dL)
ALT 166 U/L (27-101 U/L)
AST 201 U/L (17-58 U/L)
ALP 24 U/L (14-71 U/L)
GGT 0 U/L (0-4 U/L)
Cholesterol 75 mg/dL (89-258 mg/dL)
Total bilirubin 6.1 mg/dL (0-0.2 mg/dL)
Magnesium 3.4 mg/dL (1.5-2.5 mg/dL)
Creatine kinase 446 U/L (73-260 U/L).

Urinalysis: USG 1.040, pH 6, 75 mg/dL protein, no glucose,
5 mg/dL ketones, 6 mg/dL bilirubin, 250 erythrocytes/µL
hemoprotein, 0-1 RBC/HPF, 0 WBC/HPF, rare transitional
epithelial cells.
Microbiologic Testing: Real-time PCR for Mycoplasma
haemofelis, ‘Candidatus Mycoplasma haemominutum,’ and
‘Candidatus Mycoplasma turicensis’ (blood smear): Positive
for M. haemofelis DNA
Diagnosis: Feline infectious anemia (Mycoplasma haemofelis
infection)
Additional Treatment and Outcome: Treatment was
changed to doxycycline (10 mg/kg followed by 5 mL of
water PO q24h for 3 weeks). Treatment with IV crystalloid
fluids was continued and oxygen supplementation was
discontinued. Two days after admission, the PCV was 16%
and the cat became aggressive and began eating. The cat
was discharged the following day with a PCV of 18%, and
a recheck at the local veterinary clinic that included a CBC
was recommended 1 week later. The owner subsequently
elected to house Shadow indoors.
SUGGESTED READINGS
Dowers KL, Tasker S, Radecki SV, et al. Use of pradofloxacin to treat
experimentally induced Mycoplasma haemofelis infection in cats. Am
J Vet Res. 2009;70:105-111.
Tasker S, Helps CR, Day MJ, et al. Use of real-time PCR to detect and
quantify Mycoplasma haemofelis and “Candidatus Mycoplasma hae-
mominutum” DNA. J Clin Microbiol. 2003;41:439-441.
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CHAPTER 41  Hemoplasma Infections 397
Comments: This represents a typical case of hemoplasmosis in a
male neutered cat with access to the outdoors. Specific testing
for hemoplasmas with PCR was performed on blood smears
because treatment with packed RBC and antibiotics had been
initiated immediately on admission and whole blood was
not saved before treatment was instituted. Although PCR on
blood smears has lower sensitivity than when whole liquid
blood is used, in this case sufficient organisms were present
to generate a positive result, which confirmed the diagnosis
of hemoplasmosis. Because the cat was hypovolemic on
admission, parenteral antimicrobial treatment was chosen.
Subsequently, treatment with oral doxycycline was initiated.
Although continued treatment with oral enrofloxacin may
have been a suitable alternative to doxycycline, retinal
degeneration was a possible adverse effect of continued
treatment with enrofloxacin. Retesting for retroviruses was
indicated 2 months after the first test was performed at the
local veterinary clinic, as the cat may have been exposed to
retroviruses but not yet have developed positive test results
(see Chapters 21 and 22).
11. Willi B, Boretti FS, Cattori V, et al. Identification, molecular char-
acterization, and experimental transmission of a new hemoplasma
isolate from a cat with hemolytic anemia in Switzerland. J Clin
Microbiol. 2005;43:2581-2585.
12. Willi B, Tasker S, Boretti FS, et al. Phylogenetic analysis of ‘Candi-
datus Mycoplasma turicensis’ isolates from pet cats in the United
Kingdom, Australia, and South Africa, with analysis of risk factors
for infection. J Clin Microbiol. 2006;44:4430-4435.
13. Peters IR, Helps CR, Willi B, et al. The prevalence of three species
of feline haemoplasmas in samples submitted to a diagnostics ser-
vice as determined by three novel real-time duplex PCR assays. Vet
Microbiol. 2008;126:142-150.
14. Tanahara M, Miyamoto S, Nishio T, et  al. An epidemiological
survey of feline hemoplasma infection in Japan. J Vet Med Sci.
2010;72:1575-1581.
15. Sykes JE, Terry JC, Lindsay LL, et al. Prevalences of various hemo-
plasma species among cats in the United States with possible hemo-
plasmosis. J Am Vet Med Assoc. 2008;232:372-379.
16. Willi B, Boretti FS, Baumgartner C, et  al. Prevalence, risk fac-
tor analysis, and follow-up of infections caused by three feline
hemoplasma species in cats in Switzerland. J Clin Microbiol.
2006;44:961-969.
17. Nascimento NC, Santos AP, Guimaraes AM, et  al. Mycoplasma
haemocanis str. Illinois chromosome, complete genome. In:
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18. Kemming GI, Messick JB, Enders G, et al. Mycoplasma haemocanis
infection—a kennel disease? Comp Med. 2004;54:404-409.
19. Sykes JE, unpublished data, 2012.
20. André MR, Adania CH, Allegretti SM, et al. Hemoplasmas in wild
canids and felids in Brazil. J Zoo Wildl Med. 2011;42:342-347.
21. Varanat M, Maggi RG, Linder KE, et al. Molecular prevalence of
Bartonella, Babesia, and hemotropic Mycoplasma sp. in dogs with
splenic disease. J Vet Intern Med. 2011;25:1284-1291.
22. Hii SF, Kopp SR, Thompson MF, et al. Canine vector-borne dis-
ease pathogens in dogs from south-east Queensland and north-east
Northern Territory. Aust Vet J. 2012;90(4):130-135.
23. Woods JE, Brewer MM, Hawley JR, et  al. Evaluation of experi-
mental transmission of ‘Candidatus Mycoplasma haemominutum’
and Mycoplasma haemofelis by Ctenocephalides felis to cats. Am J
Vet Res. 2005;66:1008-1012.
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24. Seneviratna P, Weerasinghe, Ariyadasa S. Transmission of Haemo-
bartonella canis by the dog tick, Rhipicephalus sanguineus. Res Vet
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25. Museux K, Boretti FS, Willi B, et al. In vivo transmission studies of
‘Candidatus Mycoplasma turicensis’ in the domestic cat. Vet Res.
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26. Lumb WV. Haemobartonellosis in the dog. In: 8th Gaines Veteri-
nary Symposium 1958;15–16.
27. Maede Y. Studies on feline haemobartonellosis. IV. Lifespan of
erythrocytes of cats infected with Haemobartonella felis. Nihon
Juigaku Zasshi. 1975;37:269-272.
28. Maede Y, Hata R. Studies on feline haemobartonellosis. II. The
mechanism of anemia produced by infection with Haemobarton-
ella felis. Nihon Juigaku Zasshi. 1975;37:49-54.
29. Zulty JC, Kociba GJ. Cold agglutinins in cats with haemobartonel-
losis. J Am Vet Med Assoc. 1990;196:907-910.
30. Foley JE, Harrus S, Poland A, et al. Molecular, clinical, and patho-
logic comparison of two distinct strains of Haemobartonella felis in
domestic cats. Am J Vet Res. 1998;59:1581-1588.
31. Tasker S, Helps CR, Day MJ, et al. Use of real-time PCR to detect
and quantify Mycoplasma haemofelis and ‘Candidatus Myco-
plasma haemominutum’ DNA. J Clin Microbiol. 2003;41:439-441.
32. Harvey DG, Gaskin JM. Feline haemobartonellosis: attempts to
induce relapses of clinical disease in chronically infected cats. J Am
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33. Harvey JW, Gaskin JM. Experimental feline haemobartonellosis.
J Am Anim Hosp Assoc. 1977;13:28.
34. Alleman AR, Pate MG, Harvey JW, et  al. Western immunob-
lot analysis of the antigens of Haemobartonella felis with sera
from experimentally infected cats. J Clin Microbiol. 1999;37:
1474-1479.
35. Sykes JE, Henn JB, Kasten RW, et  al. Bartonella henselae infec-
tion in splenectomized domestic cats previously infected with
hemotropic Mycoplasma species. Vet Immunol Immunopathol.
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36. Berent LM, Messick JB, Cooper SK. Detection of Haemobarton-
ella felis in cats with experimentally induced acute and chronic
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37. Messick JB, Berent LM, Cooper SK. Development and evaluation
of a PCR-based assay for detection of Haemobartonella felis in
cats and differentiation of H. felis from related bacteria by restric-
tion fragment length polymorphism analysis. J Clin Microbiol.
1998;36:462-466.
38. Sykes JE, Owens SD, Terry JC, et al. Use of dried blood smears for
detection of feline hemoplasmas using real-time polymerase chain
reaction. J Vet Diagn Invest. 2008;20:616-620.
39. Novacco M, Wolf-Jackel G, Riond B, et  al. Humoral immune
response to a recombinant hemoplasma antigen in experimental
‘Candidatus Mycoplasma turicensis’ infection. Vet Microbiol.
2012;157:464-470.
40. Barker EN, Helps CR, Heesom KJ, et  al. Detection of humoral
response using a recombinant heat shock protein 70, DnaK, of
Mycoplasma haemofelis in experimentally and naturally hemo-
plasma-infected cats. Clin Vaccine Immunol. 2010;17:1926-1932.
41. Dowers KL, Tasker S, Radecki SV, et al. Use of pradofloxacin to
treat experimentally induced Mycoplasma haemofelis infection in
cats. Am J Vet Res. 2009;70:105-111.
42. Tasker S, Helps CR, Day MJ, et al. Use of a Taqman PCR to deter-
mine the response of Mycoplasma haemofelis infection to antibiotic
treatment. J Microbiol Methods. 2004;56:63-71.
43. Hulme-Moir KL, Barker EN, Stonelake A, et  al. Use of real-
time quantitative polymerase chain reaction to monitor antibiotic
therapy in a dog with naturally acquired Mycoplasma haemocanis
infection. J Vet Diagn Invest. 2010;22:582-587.
44. Pitorri F, Dell’Orco M, Carmichael N, et al. Use of real-time quan-
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2012;41:493-496.
45. Tasker S, Peters IR, Mumford AD, et  al. Investigation of human
haemotropic Mycoplasma infections using a novel generic haemo-
plasma qPCR assay on blood samples and blood smears. J Med
Microbiol. 2010;59:1285-1292.
46. Steer JA, Tasker S, Barker EN, et  al. A novel hemotropic Myco-
plasma (hemoplasma) in a patient with hemolytic anemia and pyrexia.
Clin Infect Dis. 2011;53:e147-151.
47. dos Santos AP, dos Santos RP, Biondo AW, et  al. Hemoplasma
infection in HIV-positive patient. Brazil. Emerg Infect Dis.
2008;14:1922-1924.
48. Sykes JE, Lindsay LL, Maggi RG, et al. Human coinfection with Bar-
tonella henselae and two hemotropic mycoplasma variants resem-
bling Mycoplasma ovis. J Clin Microbiol. 2010;48:3782-3785.
49. Yuan CL, Liang AB, Yao CB, et  al. Prevalence of Mycoplasma
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workers in Shanghai, China. Am J Vet Res. 2009;70:890-894.
CHAPTER 42

Actinomycosis
Jane E. Sykes

Actinomycosis in outdoor dogs is often related to exposure to

Overview of Actinomycosis penetrating plant foreign bodies such as grass awns. In cats,
First Described: Actinomycosis was first described in 1877 (in actinomycosis usually follows bite wounds and so is more com-
cattle) (Otto Bollinger, Germany)1 monly diagnosed in males.18,19 In the author’s hospital, two-
thirds (11 of 17) cats with actinomycosis are male. However,
Causes: Actinomyces spp. and Arcanobacterium spp. (order
underlying retrovirus infection is uncommonly present and does
Actinomycetales, family Actinomycetaceae)
not appear to predispose to disease.
Geographic Distribution: Worldwide, but especially regions Because Actinomyces species are difficult to culture and are
where plant awns are prevalent susceptible to antimicrobial drugs that are often used empirically,
Mode of Transmission: Opportunistic invasion of commensal the prevalence of actinomycosis in dogs and cats is probably
bacteria following disruption of cutaneous or mucosal underestimated.
barriers
Clinical Features
Major Clinical Signs: Subcutaneous masses and draining
skin lesions (cervicofacial and cutaneous-subcutaneous Signs and Their Pathogenesis
disease); pulmonary nodules, masses, and/or effusion
After ingestion or inhalation, plant awns become contaminated
with cough and tachypnea (thoracic disease); abdominal
with Actinomyces spp. and other bacteria from the oropharynx,
effusion or masses (abdominal disease); thoracolumbar
and then migrate to various sites and act as a nidus of infection.
pain or pelvic limb paresis/paralysis (retroperitoneal dis-
Alternatively, organisms can be introduced into tissues at the
ease); rarely neurologic signs due to meningitis or brain
time of a bite wound injury. The latter is the most common route
abscesses (central nervous system disease).
of infection in cats, and can manifest as pyothorax, peritonitis, or
Differential Diagnoses: Mycobacterial infections, streptomyco- cellulitis.18,19 Other co-infecting aerobic and anaerobic bacteria
sis, nocardiosis, bartonellosis, fungal infections, neoplasia
Human Health Significance: Direct transmission of disease from
animals to humans does not occur, but humans have devel- BOX 42-1
oped actinomycosis after bites from healthy dogs or cats.
Actinomyces and Arcanobacterium Species Isolated from
Dogs and Cats with Actinomycosis
Etiology and Epidemiology
Dogs
Actinomycosis is caused by anaerobic or microaerophilic, Actinomyces viscosus2-4
filamentous, gram-positive bacteria that belong to the genus Actinomyces bowdenii5
Actinomyces (and to a lesser extent, the related genus Arcano- Actinomyces hordeovulneris6
bacterium). These organisms are normal inhabitants of mucous Actinomyces canis7
membranes, especially of the oropharynx, but also the genital Actinomyces odontolyticus8
and gastrointestinal tracts, and cause opportunistic infections. Actinomyces catuli9
Together with other oral commensal bacteria, actinomycetes Actinomyces turicensis10
colonize the periodontal mucosal surfaces and adhere to the Actinomyces hyovaginalis11
tooth surface to form plaque. Numerous Actinomyces species Actinomyces urogenitalis11
have been cultured from the mucous membranes, dental plaque, Arcanobacterium pyogenes12
and saliva of healthy dogs and cats. Organisms isolated from
ill dogs and cats with actinomycosis are shown in Box 42-1.2-15 Cats
Usually, actinomycosis develops when Actinomyces spp. Actinomyces viscosus13,14
are inoculated into tissues along with other bacteria, often as Actinomyces bowdenii5
a result of a deeply penetrating wound or foreign body migra- Actinomyces hordeovulneris11
tion. Young adult to middle-age large-breed dogs that have out- Actinomyces meyeri15
door access are often affected, especially retriever and hunting Actinomyces odontolyticus14
breeds.16,17 There is no clear sex predisposition, and the median Arcanobacterium pyogenes12
age of dogs with actinomycosis is approximately 5 years.18

399
400 SECTION 2  Bacterial Diseases
from the oral cavity or intestinal tract (“companion microbes”)
undermine normal host defenses and reduce oxygen tension,
which allows Actinomyces spp. to persist. Actinomyces spp. that
possess fimbriae can bind to specific cell surface receptors on
other bacteria, especially streptococci. This bacterial co-aggrega-
tion inhibits the ability of neutrophils to phagocytize the organ-
isms.20 Dense colonies of Actinomyces spp. form, and these,
together with other associated bacteria, are surrounded by neu-
trophils, macrophages, and plasma cells, with slowly progressive
FIGURE 42-1  Abdominal fluid cytology from a 3-year-old male neutered domestic
longhair cat with a 1-week history of progressive lethargy and anorexia, ascites, hyper-
bilirubinemia, and hypoproteinemia. Abdominal fluid analysis revealed a total protein of
2.2 g/dL, and 75,500 nucleated cells/µL, with 92% neutrophils and 8% large mononuclear
cells. High numbers of moderately degenerative neutrophils were present with low num-
bers of macrophages. Moderate numbers of filamentous beaded bacteria were present
both in the background and within phagocytes (arrows). Actinomyces spp. and Fusobacte-
rium nucleatum were isolated from the peritoneal fluid. Necropsy revealed severe, chronic
peritonitis with intralesional filamentous bacteria.
A B
FIGURE 42-2  Cervicofacial actinomycosis in an 8-year-old female spayed Labrador retriever with slowly progressive, left-sided facial swelling. Skull radiograph (A) and computed
tomographic scan (B). There was severe, regional soft tissue swelling over the left side of the face, which involved the left masseter muscle and zygomatic salivary glands and extended into
the retrobulbar space, with secondary exophthalmos. Multiple premolars and molars of the left maxillary arcade were missing. Biopsy of the lesion revealed severe, diffuse, pyogranulo-
matous cellulitis with intralesional gram-positive, acid-fast negative bacilli. Culture yielded Actinomyces hordeovulneris (based on 16S rRNA gene sequencing), Pasteurella multocida subsp.
multocida, Neisseria canis (based on 16S rRNA gene sequencing) and an Eikenella-like organism.
pyogranulomatous inflammation (Figure 42-1). Large aggre-
gations of organisms form “sulfur granules,” which are tan to
yellow colonies of actinomycetes, which can be microscopic or
visible grossly. Proteolytic enzymes from the associated bacteria,
macrophages, and degranulated neutrophils destroy connective
tissue, which facilitates extension of the disease through normal
tissue planes. Less often, organisms spread hematogenously to
distant sites. In some cases, the inflammatory reaction is accom-
panied by mass formation and extensive fibrosis. The center
of the lesions can eventually suppurate and soften, or draining
tracts may develop. The tracts can close and reappear over weeks
to months as infection gradually spreads through tissues.
The most common clinical forms of actinomycosis in cats
and dogs involve the cervicofacial region, thorax, abdomen,
and subcutaneous tissue, but central nervous system (CNS)
infections (meningitis and meningoencephalitis) and ocular
infections (keratitis and endophthalmitis) can also occur.21,22
Cervicofacial actinomycosis can follow bite wounds, perfora-
tion of the oropharynx by a foreign body, or chronic periodon-
tal disease. The mandibular, submandibular, and ventral or
lateral cervical areas are most frequently affected, but infections
that involve the face, retrobulbar space, and temporal area also
occur (Figure 42-2, A and B). Cutaneous-subcutaneous actino-
mycosis typically involves the lateral thoracic wall, flank region,
and occasionally the limbs. Lesions on the thoracic or abdomi-
nal walls may represent extensions of thoracic, abdominal, or
retroperitoneal actinomycosis.
Thoracic actinomycosis may be limited to the lung paren-
chyma but can involve other thoracic structures, such as the
mediastinum, pleura, pericardium, and thoracic wall. It usually
follows aspiration of oropharyngeal material, often together
with a contaminated grass awn. Alternative routes of thoracic
infection include mediastinal involvement after esophageal
perforation or direct extension of subcutaneous or abdomi-
nal disease. Clinical signs include cough and less commonly
hemoptysis, tachypnea, respiratory distress, and subcutaneous
 CHAPTER 42  Actinomycosis 401

soft tissue masses or draining skin lesions on the thoracic wall.
Sometimes there is a history of neck pain, gagging, or hypersali-
vation before the onset of tachypnea, possibly due to passage of
a penetrating foreign body.
Abdominal actinomycosis develops when ingested foreign
bodies penetrate the gastrointestinal tract, which leads to the
formation of intra-abdominal mass lesions and ascites.2,16,23
Abdominal involvement may also follow direct extension from
subcutaneous tissues or hematogenous spread of the organism
to abdominal organs such as the liver. Although dogs are more
often affected than cats, intra-abdominal actinomycotic masses
can occur in cats.24,25 Retroperitoneal actinomycosis in dogs
often follows the migration of plant awns through the lung and
up the crus of the diaphragm to its dorsal attachment. Initially,
fever of unknown origin may be the only clinical sign. Ulti-
mately, progression can lead to osteomyelitis and compression
fractures of the vertebral bodies, with spinal pain and paresis or
paralysis of the pelvic limbs (Figure 42-3).3,26,27
Although rare, actinomycosis of the brain and meninges can
also occur in dogs (Figure 42-4).28-30 Brain abscesses may follow
hematogenous dissemination from a distant site, or meningitis
may result from extension of infection from an adjacent site,
such as the middle ear.29,31 In humans, risk factors for CNS
actinomycosis include dental disease or tooth extraction, head
trauma, gastrointestinal tract surgery, and chronic otitis, mas-
toiditis, or sinusitis. CNS actinomycosis has been reported in
dogs in association with concurrent otitis media/interna30 and
head trauma,28 and in cats in association with retrobulbar
abscesses31 and cutaneous abscesses of the tail base.13,32
Rarely, Actinomyces infections of the urinary bladder,12,33
gallbladder,34,35 and heart valve4,10,36,37 have been recognized in
dogs, cats, and human patients.
Physical Examination Findings
Lesions in dogs and cats with cervicofacial or cutaneous-sub-
cutaneous actinomycosis may fluctuant or firm, indurated,
have draining sinuses, and rarely are ulcerated. Pain and fever
are variable. Thoracic or abdominal actinomycosis is often
accompanied by a thin body condition and fever. Dogs and cats
with thoracic actinomycosis may show tachypnea or respiratory
distress, and thoracic auscultation may reveal decreased lung
sounds if pyothorax or intrathoracic mass lesions are present.
Masses and/or ascites may be detected on abdominal palpation
of dogs with abdominal actinomycosis. Dogs with retroperito-
neal actinomycosis can show pain on palpation of the abdomen
or spine; signs of pelvic limb paralysis or paresis may be also
present, such as pelvic limb ataxia, delayed or absent placing
reactions, increased segmental reflexes, and loss of pain sensa-
tion. Neurologic signs in dogs with Actinomyces meningitis or
encephalitis include altered behavior, decreased consciousness,
cervical pain, vision loss, ataxia, tetraparesis, and seizures.
FIGURE 42-4  Necropsy findings in a 1-year-old male neutered Yorkshire terrier with
suspected CNS actinomycosis. Three weeks previously, surgery to correct an extrahepatic
portosystemic shunt and remove a urate cystolith had been performed. The dog’s appetite
and activity level did not improve after the surgery, and for the week before the dog was
reexamined, disorientation and weakness was noted. The dog deteriorated rapidly after it
was hospitalized, and euthanasia was performed. Three separate abscesses were found in
the left cerebral cortex. Histopathology revealed neutrophilic inflammation, hemorrhage,
and granular mats of material that contained gram-positive, acid-fast negative branching
bacteria (consistent with Actinomyces spp.), as well as a dense population of gram-nega-
tive, Giemsa-positive cocci. (Courtesy University of California, Davis, Veterinary Anatomic
Pathology Service.)

FIGURE 42-3  Left lateral spinal radiograph from a 3-year-old female Border collie with retroperitoneal actinomycosis. The dog was seen for a 3-day history of lethargy, fever, abdomi-
nal pain, and progressive pelvic limb paresis. Severe, smoothly marginated ventral spondylosis bridges the L1-L4 space. There is also marked irregular osteolysis of the 2nd, 3rd, and 4th
lumbar vertebrae. There is loss of visualization, narrowing, and irregularity of the intervertebral disc spaces at L2-3 and L3-4. There is marked loss of detail within the retroperitoneal space,
and the colon is displaced slightly ventrally. Irregular mineral foreign bodies are present in the stomach and colon.
402 SECTION 2  Bacterial Diseases
Diagnosis
In the human literature, actinomycosis has been referred to
as the “most misdiagnosed disease.”38 Fibrous masses are fre-
quently mistaken for neoplastic lesions, although reports of
Actinomyces spp. infections that complicate neoplasia exist in
human medicine.39,40 Aspiration of firm lesions is often unre-
warding, organisms are often difficult to isolate from lesions,
and short periods of antimicrobial drug treatment are ineffec-
tive, which all serve to further mislead the clinician to a diag-
nosis of neoplasia. Actinomycosis should be considered on
the list of differential diagnosis in any animal with a history
of penetrating plant awn foreign bodies, draining skin lesions,
chronic fibrous masses, persistent bite wounds, pyothorax,
retroperitoneal abscesses, or focal neurologic signs, especially
when young-adult to middle-aged dogs are affected. Ultimately,
diagnosis is based on clinical signs, identification of organisms
with typical morphology with cytology or histopathology, and
culture.
Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
Animals with extensive, chronic actinomycosis may have mild
to moderate nonregenerative anemia, leukocytosis with a mild
to moderate left shift and monocytosis, hypoalbuminemia, and
hyperglobulinemia, which may be marked. Dogs with body cav-
ity effusions may be hypoglycemic. There are usually no specific
urinalysis findings.
A C
FIGURE 42-5  Imaging findings in a 1-year-old female spayed Labrador retriever with systemic actinomycosis secondary to grass awn migration. A, Dorsoventral thoracic radiograph.
Patchy diffuse interstitial opacities were present in all lung fields. B, Ultrasound image of the liver. Within the left liver, there were focal, ill-defined heteroechoic or hypoechoic nodules. One
of these areas (shown) had a hyperechoic rim with echogenic luminal contents. The nodules were consistent with multifocal hepatic abscesses. C, Ultrasound image of the thoracic wall.
A linear plant awn foreign body was identified (arrow).
Cytologic Examination of Body Fluids
Cytologic examination of aspirates of abscesses, effusions, or
CSF from animals with actinomycosis typically reveals a suppu-
rative to pyogranulomatous inflammatory response (>75% neu-
trophils). Total protein in pleural fluid is generally greater than
3.0 g/dL, with erythrocyte and nucleated cell counts often greater
than 70,000 cells/µL. CSF from dogs and cats with cerebral acti-
nomycosis can grossly resemble pus. Aspirates of firm masses
may yield only a small amount of blood. Sulfur granules may
be visible in effusion fluid or purulent material from cutaneous
lesions, grossly as white to tan to gray granules, or microscopi-
cally as dense clusters of organisms. When bacteria are visual-
ized, they may be filamentous rods suggestive of Actinomyces
spp., or “companion” microorganisms. Actinomycetes are gram-
positive, non-acid-fast filamentous organisms that are occasion-
ally branched. The filaments are less than 1 µm wide, vary in
length, and can stain irregularly, producing a beaded appearance
(see Figure 42-1). Nocardia spp., Corynebacterium spp., and
Mycobacterium spp. can be confused with Actinomyces species.
Diagnostic Imaging
Plain Radiography
Radiographs of cervicofacial or cutaneous-subcutaneous lesions
may show evidence of adjacent osteomyelitis and/or periosteal
new bone formation. Pulmonary involvement may manifest as
interstitial or alveolar infiltrates, sometimes with consolidation
(Figure 42-5, A). Air bronchograms may be seen within pulmo-
nary mass lesions, which suggests the presence of a nonneoplastic

process. Other variable findings include pleural thickening, pleu-
ral effusion, widening of the mediastinum, pleural mass lesions,
enlargement of the cardiac silhouette (with pericardial involve-
ment), and periosteal new bone formation or osteomyelitis
involving adjacent ribs, vertebral bodies, or sternebrae. Dogs
with retroperitoneal actinomycosis may have periosteal new
bone formation on the ventral aspects of two or three adjacent
vertebral bodies (usually T13 through L4) on abdominal radi-
ography; involvement of disc spaces is uncommon (see Figure
42-3). Abdominal actinomycosis may be characterized by loss of
abdominal detail due to peritoneal effusion and intra-abdominal
mass lesions.
Sonographic Findings
Ultrasonography in dogs with actinomycosis can be useful to
identify linear grass awn foreign bodies (see Figure 42-5).41,42
Sonographic abnormalities in dogs with abdominal disease
include variable amounts of flocculent ascites fluid, abdominal
lymphadenopathy, and nodular or mass lesions that incorporate
or displace adjacent structures. New bone formation may be
detected on interrogation of the ventral aspects of the vertebral
bodies in dogs with retroperitoneal disease.
Advanced Imaging
For animals with CNS involvement, MRI may reveal meningeal
thickening, contrast enhancement, and/or intraparenchymal
contrast-enhancing mass lesions.18 MRI may also be useful for
detection of grass awn foreign bodies. Advanced imaging allows
assessment of the extent of actinomycosis (see Figure 42-2),
which can assist with surgical planning.
Microbiologic Tests
Diagnostic assays available for Actinomyces infection in dogs
and cats are shown in Table 42-1.
Isolation and Identification
Because Actinomyces is a commensal of the oral cavity, it is
commonly swallowed, inhaled, and transferred by licking; there-
fore, culture of the organism from the airways, gastrointestinal
tract, or skin does not necessarily constitute infection. Consis-
tent cytologic or histopathologic findings (pyogranulomatous
TABLE 42-1
Diagnostic Assays Available for Actinomyces Infection in Dogs and Cats
Assay Specimen Type Target
Culture Aspirates of purulent material Actinomyces spp.
(e.g., abscesses, pleural effu-
sion), whole blood, CSF, tissue
specimens obtained by biopsy
or at necropsy (e.g., subcutane-
ous tissue, heart valve, lung
tissue), sulfur granules
Cytology or histopa- Aspirates or tissue specimens Actinomyces spp.
thology with organ- organisms
ism detection using
special stains
CHAPTER 42  Actinomycosis 403
inflammation with or without the presence of filamentous bac-
teria) and the presence of companion microorganisms support
the diagnosis of actinomycosis.
Specimens for culture can be collected through fine-needle
aspiration or, if possible, biopsy. Because Actinomyces species
are susceptible to many antimicrobial drugs, treatment of ani-
mals before obtaining specimens for culture can also prevent
recovery of the organisms. Culture may be negative or yield only
“companion” microorganisms, which can obscure the presence
of Actinomyces spp. As a result, diagnosis is often based only
on cytologic or histologic identification of the organism in speci-
mens from animals with appropriate clinical signs.
Most Actinomyces spp. that cause disease in dogs and
cats are facultative anaerobes, but a few (A. bovis, A. israelii,
A. meyeri) are obligate anaerobes.5,7,9,43 The facultative anaer-
obes can grow under aerobic conditions, and some, such as
A. viscosus, grow best in these conditions. In addition, “com-
panion” microorganisms are often obligate anaerobes or other
aerobes. Therefore, both aerobic and anaerobic cultures should
be requested when an Actinomyces spp. infection is suspected,
and the laboratory should be notified that Actinomyces spp.
may be present. Tissue specimens, aspirates of pus, or sulfur
granules are ideal specimens for anaerobic culture.
Visible growth of Actinomyces spp. can occur within 48 hours
but usually requires 5 to 7 days. It may be necessary to hold
plates 2 to 4 weeks. Species identification using traditional bio-
chemical tests is difficult. PCR amplification and DNA sequence
analysis of the 16S rRNA gene may be necessary for precise spe-
cies identification. In the future, matrix-assisted laser desorption/
ionization-time of flight (MALDI-TOF) mass spectrometry may
prove most useful for identification of Actinomyces species (see
Chapter 3).44
In addition to actinomycetes, one to five other associated
bacteria are often recovered from properly handled specimens.
The most commonly isolated organisms are resident flora of the
oral cavity or intestinal tract and include the anaerobes Fuso-
bacterium, Peptostreptococcus, Prevotella, or Bacteroides spp.;
and Pasteurella multocida, Escherichia coli, and Streptococcus
spp. Occasionally, pure cultures of Actinomyces spp. are iso-
lated, but this does not exclude the concurrent presence of other
organisms, especially strict anaerobes.
Performance
Some organisms require anaerobic conditions.
Requires several weeks’ incubation, and over-
growth by companion bacteria may occur.
Because Actinomyces spp. are mucosal com-
mensal bacteria, isolation from contaminated
sites does not imply disease causation. PCR
and sequencing may be required to identify
the species present.
Organisms are gram positive, do not take up
acid-fast stain, and are often accompanied by
other bacteria and sulfur granules. Definitive
diagnosis requires culture because Nocardia
can resemble Actinomyces spp.
404 SECTION 2  Bacterial Diseases

A B
FIGURE 42-6  Histopathology of a biopsy specimen from the xiphoid of a 10-year-old intact male English pointer dog with actinomycosis due to a migrating plant awn foreign body.
The dog had a 1-month history of a ventral thoracic wall mass. A, Dense, granular mats of branching bacteria were present in a background of extensive granulation tissue and embedded
plant material (not shown). H&E stain. B, Brown and Benn (gram) stain revealed colonies of gram-positive, filamentous, branching bacteria. Actinomyces bowdenii (based on 16S rRNA
gene sequencing), Pasteurella canis, Porphyromonas canoris, Propionibacterium acnes, a Bacteroides gingivalis–like organism, and a Bacteroides ureolyticus–like organism were isolated
from the lesion.

Molecular Diagnosis Using the Polymerase Chain Reaction
Specific PCR assays for Actinomyces spp. have been developed
and used for diagnosis of human actinomycosis45 but are not
commercially available for veterinary diagnosis.
Pathologic Findings
Gross lesions in animals with actinomycosis often consist of
one or more poorly defined, indurated masses of the subcu-
taneous tissues, thoracic cavity, lungs, abdominal cavity, or
retroperitoneal space, which incorporate adjacent structures.
Masses may contain pockets of a reddish-brown exudate.
Fistulas, plant material, and sulfur granules may be found.
Abdominal or thoracic effusions are often reddish-brown and
can contain sulfur granules. Animals with thoracic and abdom-
inal infections may also have a diffuse, red, velvety to granular
thickening of the pleura or peritoneum and omentum. Rarely,
abscesses are present in the brain, or purulent meningitis is
identified.
Histopathology of affected tissues reveals abscesses with a
core of neutrophils encapsulated by granulation tissue that con-
tains macrophages, plasma cells, and lymphocytes in a dense,
fibrous tissue matrix. When present, sulfur granules are gen-
erally in the center of microabscesses, but multiple tissue sec-
tions may be needed to find them. In tissue sections stained
with hematoxylin and eosin, the granules are round, oval, or
scalloped amphophilic solid masses (Figure 42-6). They vary
in size from 30 to 3000 µm in diameter and often are rimmed
by partially confluent radiating eosinophilic club-shaped struc-
tures collectively known as the Splendore-Hoeppli phenom-
enon. This phenomenon can also occur with zygomycosis,
sporotrichosis, parasitic infections, foreign body reactions, and
hypereosinophilic syndrome.46,47 Antigen-antibody complexes,
complement, and major basic protein of eosinophils have been
detected within the Splendore-Hoeppli phenomenon. Special
stains (Gram stains, Giemsa and silver stains) are required to
stain Actinomyces spp. and reveal clumps of tangled, intermit-
tently branched, thin (<1 µm) filaments (see Figure 42-6, B).
Other nonfilamentous bacteria and/or plant material may also
be present. Actinomyces are not acid fast. With the rare excep-
tion of some Nocardia spp. that are not acid fast, other fungi
and bacteria that produce tissue granules can be distinguished
from Actinomyces based on their staining and morphologic
properties.
Treatment and Prognosis
Treatment of actinomycosis usually requires prolonged antimi-
crobial drug treatment. Penicillin is the antimicrobial drug of
choice because isolates are uniformly susceptible to penicillins
(Table 42-2). High-dose therapy for weeks to months is gener-
ally required for animals with chronic infections and extensive
fibrosis. In human patients with actinomycosis, high doses of
penicillin are given parenterally for 2 to 6 weeks, followed by
oral therapy with amoxicillin for 6 to 12 months.48 If the animal
is stable clinically, oral therapy can be tried from the outset.8,49
Treatment must be extended for weeks to months beyond reso-
lution of measurable disease to prevent relapse; in some cases,
treatment for longer than a year may be required.8,16 However,
when actinomycosis is associated with foreign body migration,
shorter durations of treatment may be possible if the foreign
body is removed, drainage is established, and extensive scar
tissue has not formed. Alternative antimicrobial drug choices
include clindamycin, doxycycline, chloramphenicol, carbap-
enems, and ceftriaxone.48 It is recommended that cephalexin,
metronidazole, and aminoglycosides be avoided for treatment
of human actinomycosis.48 Infections associated with com-
panion microbes also usually resolve with penicillin, but on
occasion they require broader spectrum antibiotics during the
initial treatment period followed by long-term administration
of penicillin.
Excepting when foreign body material is present (Figure 42-7),
surgery has a controversial role in the treatment of actinomyco-
sis, but may be required if there is extensive fibrous tissue for-
mation or large accumulations of purulent material to facilitate

TABLE 42-2
Medications That Could Be Used to Treat Actinomyces spp. Infections in Dogs and Cats with Actinomycosis
Drug Dose
Penicillin G 100,000 U/kg
Ampicillin sodium 20
Amoxicillin 20
Clindamycin* 11
Doxycycline† 5-10
*For IV administration of clindamycin phosphate, see Chapter 8.
†10 mg/kg q12h preferred if tolerated without gastrointestinal adverse effects. Doxycycline hyclate or clindamycin should always be administered
with a bolus of water to avoid esophagitis.
FIGURE 42-7  One-year-old female spayed Labrador retriever with systemic actino-
mycosis secondary to a plant awn foreign body after surgical removal of the plant awn and
placement of a grenade drain.
drug penetration (see Chapter 87 for information on manage-
ment of pyothorax). In human medicine, an initial attempt to
control disease with aggressive medical therapy alone has been
suggested, with surgical therapy if the response to treatment is
inadequate.48 In animals with pulmonary abscesses, removal of
affected lung lobes may be required. Fibrotic lesions are often
extensively vascularized and may obliterate tissue planes, which
complicates dissection. In dogs with solitary masses that involve
the thoracic and abdominal walls, radical surgical excision is
often curative, but repeat surgeries may be needed. An initial
CASE EXAMPLE
Signalment: “Penny”, a 1-year-old, female spayed Labrador
retriever from Herald in northern California
History: Penny was evaluated by the University of California,
Davis, veterinary emergency service for a 1-week history of
progressive inappetence, weight loss, lethargy, and fever.
Two days before she was evaluated, Penny vomited up some
kibble, and had not eaten anything since. The owner reported
that her feces had been formed and there were no changes
CHAPTER 42  Actinomycosis 405
Route Interval (hours)
IV, IM 6-8
IV, IM, SC 6-8
PO 8
PO 12 (dogs), 24 (cats)
PO 12-24
period of antibiotic therapy may reduce the size of lesions and
improve lesion definition, which facilitates surgery followed by
continued antimicrobial drug treatment at a later date. Surgery
is also required if a nidus such as a grass awn is present.
Appropriate treatment of actinomycosis results in a cure rate
of greater than 90%.8,16,50 Cure rates with meningitis/meningo-
encephalitis and advanced thoracic or peritoneal disease with
extensive mass lesions may be lower.
Prevention
Prevention of actinomycosis involves restriction of access to
areas with extensive grass awn formation and avoidance of
fighting and biting activity between animals. When wounds
occur, they should be carefully examined for foreign material
and cleaned promptly. Plant awns found on animals’ coats
should be removed and discarded as soon as possible and the
finding of one should prompt a thorough search for others.
Public Health Aspects
No reports exist of actinomycosis being transmitted from clini-
cally infected animals to humans or other animals; however,
humans bitten by dogs, cats, or other people can develop acti-
nomycosis (see Chapter 57).51 The most common species that
infects humans is A. israelii.48 This is a commensal of the canine
oral cavity but has not been isolated from dogs or cats with
actinomycosis. Nevertheless, A. viscosus, A. odontolyticus, and
A. meyeri have been isolated from canine and feline as well as
human actinomycosis.
in thirst or urination. Earlier the same day, Penny had been
evaluated at another veterinary clinic where a grade IV/
VI left-sided cardiac murmur was auscultated and thoracic
radiographs showed a bronchoalveolar and interstitial lung
pattern. She was treated with cefazolin (27 mg/kg IV) and
furosemide (1 mg/kg SC) and referred. Penny had been fully
vaccinated for canine distemper virus, canine parvovirus,
canine adenovirus, and rabies. She had missed some doses
of heartworm prophylaxis medication recently and received
no other medications. She lived with three other healthy
406 SECTION 2  Bacterial Diseases
Labrador retrievers. She roamed on 8 acres of land, and
there was a pond on the property where she swam and was
known to eat crayfish and frogs. Her usual diet consisted of
a commercial dry puppy food. Seven months earlier, she had
been evaluated at another veterinary clinic for vomiting and
inappetence. At that time she was moderately azotemic;
an exploratory laparotomy was performed for a suspected
foreign body, but no foreign body was found.
Physical Examination:
Body Weight: 22.5 kg
General: Quiet, alert, responsive and wagging tail.
Approximately 5% dehydrated. T = 105.2°F (40.7°C), HR 160
beats/min, RR = 42 breaths/min. Pink, slightly tacky mucous
membranes, CRT < 2 s.
Integument: Full clean haircoat, no evidence of ectoparasites.
Eyes, Ears, Nose, and Throat: No clinically significant
abnormalities were noted.
Musculoskeletal: BCS 3/9. Generalized mild muscle atrophy
was present.
Cardiovascular: A grade II/VI left systolic murmur was
auscultated. Femoral pulses were strong, regular, and
synchronous.
Respiratory: Increased breath sounds were present bilaterally
and diffusely. There was a mild increase in respiratory effort.
All Other Systems: No clinically significant abnormalities were
noted.
Laboratory Findings:
CBC:
HCT 24.0% (40%-55%),
MCV 65.6 fL (65-75 fL)
MCHC 35.0 g/dL (33-36 g/dL)
WBC 29,850 cells/µL (6000-13,000 cells/µL)
Neutrophils 16,119 cells/µL (3000-10,500 cells/µL)
Lymphocytes 2985 cells/µL (1000-4000 cells/µL)
Monocytes 7761 cells/µL (150-1200 cells/µL)
Eosinophils 0 cells/µL (0-1500 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL)
Platelets 98,000 platelets/µL (150,000-400,000 platelets/µL)
MPV 20.1 fL (7-13 fL).
A few slightly toxic band neutrophils and moderate
numbers of macroplatelets were present.
Serum Chemistry Profile:
Sodium 144 mmol/L (145-154 mmol/L)
Potassium 4.3 mmol/L (3.6-5.3 mmol/L)
Chloride 114 mmol/L (108-118 mmol/L)
Bicarbonate 15 mmol/L (16-26 mmol/L)
Phosphorus 5.3 mg/dL (3.0-6.2 mg/dL)
Calcium 9.6 mg/dL (9.7-11.5 mg/dL)
BUN 22 mg/dL (5-21 mg/dL)
creatinine 0.8 mg/dL (0.3-1.2 mg/dL)
Glucose 44 mg/dL (64-123 mg/dL)
Total protein 7.8 g/dL (5.4-7.6 g/dL)
Albumin 2.5 g/dL (3.0-4.4 g/dL)
Globulin 5.3 g/dL (1.8-3.9 g/dL)
ALT 31 U/L (19-67 U/L)
AST 76 U/L (19-42 U/L)
ALP 612 U/L (21-170 U/L)
Gamma GT 5 U/L (0-6 U/L)
Cholesterol 292 mg/dL (135-361 mg/dL)
Total bilirubin 0.8 mg/dL (0-0.2 mg/dL)
Urinalysis: SGr 1.016; pH 5.0, 1+ protein (SSA), 1+ bilirubin,
2+ hemoprotein, 1+ glucose, 0-1 WBC/HPF, 0-3 RBC/HPF,
rare granular casts, many amorphous crystals, rare bilirubin
crystals, many lipid droplets
Coagulation Panel: PT 7.7 s (7.5-10.5 s), APTT 15.2 s (9-12 s),
fibrinogen 232 mg/dL (90-255 mg/dL), D-dimer 1.0-2.0 µg/
mL (0-0.25 µg/mL).
Imaging Findings:
Thoracic Radiographs (3 View): There were patchy diffuse
unstructured interstitial opacities in all lung lobes (see
Figure 42-5, A). Cardiovascular structures appeared within
normal limits. The liver was markedly enlarged.
Echocardiography: The left ventricle was eccentrically
hypertrophied. The papillary muscles had normal shape and
echogenicity. There was no enlargement of the left atrium.
There was no evidence of endocarditis. Decreased fractional
shortening (26%; reference range, approximately 34%-46%)
and increased end systolic diameter (3.9 cm; reference range,
approximately 1.8-3.5 cm) and E-point septal separation (6.8
mm) were indicative of systolic dysfunction, which could
also be appreciated subjectively. There was mild tricuspid
regurgitation and trivial pulmonic insufficiency. Conclusions:
Moderate systolic dysfunction. It was recommended that
the dog’s diet and taurine blood/plasma levels be evaluated.
It was also considered possible that the dog’s febrile state
was responsible for some of abnormalities observed on the
echocardiogram.
Abdominal Ultrasound: The liver was diffusely mottled,
particularly in the left caudal region (see Figure 42-5, B).
Within the left caudal liver, there was a focal, ill-defined
heteroechoic area approximately 1 cm in diameter. Other
smaller, ill-defined hypoechoic nodules were also present
within the left liver. One of these areas had a well-demarcated,
hyperechoic rim with irregular echogenic luminal contents
and measured approximately 0.5 cm in diameter. Multiple
large mesenteric lymph nodes were just caudal to the liver;
the largest measured 1.3 cm by 4.5 cm. There was a small
amount of anechoic free abdominal fluid. The spleen was
moderately enlarged, but the echotexture and echogenicity
appeared within normal limits. There was mild renal
pyelectasia bilaterally, but the renal papillae appeared within
normal limits.
Microbiologic Testing: Aerobic and anaerobic blood
cultures (five specimens): Negative
IFA serology for serum antibody to Ehrlichia canis, Anaplas-
ma phagocytophilum, Rickettsia rickettsii, and Babesia ca-
nis: Negative
IFA serology for serum antibody to Bartonella vinsonii subsp.
berkhoffii, Bartonella clarridgeiae, and Bartonella hense-
lae: Negative
Bartonella culture (whole blood): Negative
Serology for Coccidioides serum antibodies: Negative
Cytologic Findings:
Splenic Aspirate (Ultrasound-Guided): Two smears were
examined that contained a large amount of blood in
the background and were highly cellular. Several small
clumps of splenic stromal cells were noted. A large
heterogeneous population of lymphocytes was found,
with a moderate increase in plasma cells. A moderate
number of hematopoietic cells was also present, including

megakaryocytes, myeloid, and erythroid precursors.
Interpretation: Moderate reactive lymphoid hyperplasia and
extramedullary hematopoiesis.
Liver Aspirate (Ultrasound-Guided): Smears contained a
diffusely stippled background with abundant nuclear debris
and were highly cellular. A large population of mildly to
markedly degenerate neutrophils were observed. Degenerate
cells ranged from highly pyknotic to severely karyolytic.
A few neutrophils contained small intracellular bacterial
rods. Interpretation: Marked suppurative inflammation with
bacterial sepsis.
Further Microbiologic Testing:
Aerobic and Anaerobic Culture (Ultrasound-Guided Liver
Aspirate): Small numbers of suspect Actinomyces spp.
No anaerobes cultured. Partial 16S rRNA gene PCR and
sequence analysis revealed 99% identity to Actinomyces
bowdenii.
Diagnosis: Systemic A. bowdenii infection with hepatic
involvement
Treatment: Immediately on admission, Penny was treated
with IV lactated Ringer’s solution with 20 mEq/L KCl and 5%
dextrose at 75 mL/hr. After the specimens for blood culture
were collected, treatment with enrofloxacin (10 mg/kg
IV q24h) and ampicillin (22 mg/kg IV q8h) was initiated.
Within 24 hours, the dog’s rectal temperature had decreased
to 101.3°F (38.5°C), the blood glucose had increased from
41 mg/dL to 107 mg/dL, and Penny began eating. The liver
aspirate was submitted for culture on day 2 of hospitalization.
A recheck abdominal ultrasound examination on day 3
showed progressive abscessation of the left liver and further
enlargement of the gastrohepatic lymph nodes. By day
4 of hospitalization, growth of an organism resembling
an Actinomyces sp. was reported. On day 5, antimicrobial
drug treatment was changed to clavulanic acid–amoxicillin
(17 mg/kg q8h). However, later in the day, Penny’s temperature
increased to 103.6°F (39.8°C), and so the initial antimicrobial
drug prescription was reinstated. The dog’s hematocrit had
also dropped to 19%. Thoracic radiographs were repeated and
showed almost complete resolution of the patchy pulmonary
infiltrates. A follow-up echocardiogram was unchanged.
Abdominal ultrasound examination was again repeated and
SUGGESTED READINGS
Barnes LD, Grahn BH. Actinomyces endophthalmitis and pneumonia in
a dog. Can Vet J. 2007;48:1155-1158.
Edwards DF, Nyland TG, Weigel JP. Thoracic, abdominal, and verte-
bral actinomycosis. Diagnosis and long-term therapy in three dogs.
J Vet Intern Med. 1988;2:184-191.
Kirpensteijn J, Fingland RB. Cutaneous actinomycosis and nocardiosis in
dogs: 48 cases (1980-1990). J Am Vet Med Assoc. 1992;201:917-920.
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Wissensch. 1877;15:481-485.
2. Georg LK, Brown JM, Baker HJ, et al. Actinomyces ­viscosus as an
agent of actinomycosis in the dog. Am J Vet Res. 1972;33:1457-1470.
3. Davenport AA, Carter GR, Schirmer RG. Canine actinomycosis
due to Actinomyces viscosus: report of six cases. Vet Med Small
Anim Clin. 1974;69:1442, 1444-1447.
CHAPTER 42  Actinomycosis 407
showed persistent hypoechogenicity of the left liver lobe,
but during the procedure a hyperechoic lesion that was
consistent with a plant awn foreign body was discovered
in the subcutaneous tissue in a caudoventral intercostal
space. On day 6, surgery was performed to remove the
plant awn. Packed red cells were administered during
the procedure, and a 16F thoracostomy tube was placed
because the thoracic cavity was entered during the search
for the foxtail, which, with the assistance of intraoperative
ultrasound examination, was eventually located under the
13th rib just outside the pleural space. Sulfur granules were
found embedded in the tissue during surgery. A closed
suction grenade drain was also placed (see Figure 42-7). The
thoracostomy tube was removed later in the day. Treatment
with enrofloxacin and ampicillin was continued, as well as
with oxymorphone (0.04 mg/kg SC q6h) to control pain.
On day 7, Penny’s temperature was 100.7°F (38.2°C), and
the drain was removed. Aerobic and anaerobic culture of
the foxtail revealed small numbers of penicillin-susceptible
Enterococcus faecalis and an organism resembling a
Streptomyces sp. No anaerobes were cultured. Penny was
discharged on day 8 with instructions to continue treatment
with clavulanic acid–amoxicillin for an additional month. The
dog had no further clinical signs of illness.
Comments: This is an unusual case of disseminated A. bowdenii
infection that led to hematogenous pneumonia and hepatic
abscessation. Although there was an initial defervescence
with antimicrobial drug treatment, anemia and pyrexia
persisted, which led to a search for an abscess that might
require drainage or a plant awn. Fortunately, a plant awn
was ultimately identified with ultrasound and removed, after
which the dog’s illness resolved completely. E. faecalis was
isolated from the plant awn and represented a “companion
microbe,” but whether dissemination of this organism
occurred was unclear. The Streptomyces sp. may have been a
contaminant, or the Actinomyces sp. that was misidentified.
The owner was warned that additional plant awns might
still be present. The cause of the cardiomyopathy was not
determined; plasma and whole blood taurine concentrations
were within normal limits, and follow-up examination was
not performed.
4. Meurs KM, Heaney AM, Atkins CE, et  al. Comparison of poly-
merase chain reaction with bacterial 16S primers to blood culture
to identify bacteremia in dogs with suspected bacterial endocardi-
tis. J Vet Intern Med. 2011;25:959-962.
5. Pascual C, Foster G, Falsen E, et al. Actinomyces bowdenii sp. nov.,
isolated from canine and feline clinical specimens. Int J Syst Bacte-
riol. 1999;49(Pt 4):1873-1877.
6. Buchanan AM, Scott JL. Actinomyces hordeovulneris, a canine
pathogen that produces L-phase variants spontaneously with coin-
cident calcium deposition. Am J Vet Res. 1984;45:2552-2560.
7. Hoyles L, Falsen E, Foster G, et  al. Actinomyces coleocanis
sp. nov., from the vagina of a dog. Int J Syst Evol Microbiol.
2002;52:1201-1203.
8. Edwards DF, Nyland TG, Weigel JP. Thoracic, abdominal, and
vertebral actinomycosis. Diagnosis and long-term therapy in three
dogs. J Vet Intern Med. 1988;2:184-191.
9. Hoyles L, Falsen E, Pascual C, et al. Actinomyces catuli sp. nov.,
from dogs. Int J Syst Evol Microbiol. 2001;51:679-682.
408 SECTION 2  Bacterial Diseases
10. Junius G, Bavegems V, Stalpaert M, et  al. Mitral valve endo-
carditis in a Labrador retriever caused by an actinomyces spe-
cies identified as Actinomyces turicensis. J Vet Intern Med.
2004;18:899-901.
11. Sykes JE, Unpublished observations, 2012.
12. Billington SJ, Post KW, Jost BH. Isolation of Arcanobacterium
(Actinomyces) pyogenes from cases of feline otitis externa and
canine cystitis. J Vet Diagn Invest. 2002;14:159-162.
13. Bestetti G, Bühlman V, Nicolet J, et  al. Paraplegia due to Acti-
nomyces viscosus infection in a cat. Acta Neuropathol (Berl).
1977;39:231-235.
14. Love DN, Jones RF, Bailey M, et al. Isolation and characterisation
of bacteria from abscesses in the subcutis of cats. J Med Microbiol.
1979;12:207-212.
15. Love DN, Jones RF, Bailey M, et al. Isolation and characterisation
of bacteria from pyothorax (empyaemia) in cats. Vet Microbiol.
1982;7:455-461.
16. Hardie EM, Barsanti JA. Treatment of canine actinomycosis. J Am
Vet Med Assoc. 1982;180:537-541.
17. Kirpensteijn J, Fingland RB. Cutaneous actinomycosis and nocar-
diosis in dogs: 48 cases (1980-1990). J Am Vet Med Assoc.
1992;201:917-920.
18. Sykes JE. Actinomycosis and nocardiosis. In: Greene CE, ed. Infec-
tious Diseases of the Dog and Cat. 4th ed. St. Louis: Elsevier Saun-
ders; 2012:484-495.
19. Love DN, Jones RF, Bailey M, et al. Bacteria isolated from subcu-
taneous abscesses in cats. Aust Vet Pract. 1978;8:87-90.
20. Ochiai K, Kurita-Ochiai T, Kamino Y, et  al. Effect of co-
aggregation on the pathogenicity of oral bacteria. J Med Microbiol.
1993;39:183-190.
21. Ledbetter EC, Scarlett JM. Isolation of obligate anaerobic bacte-
ria from ulcerative keratitis in domestic animals. Vet Ophthalmol.
2008;11:114-122.
22. Barnes LD, Grahn BH. Actinomyces endophthalmitis and pneumo-
nia in a dog. Can Vet J. 2007;48:1155-1158.
23. Chastain CB, Grier RL, Hogle RM, et al. Actinomycotic peritonitis
in a dog. J Am Vet Med Assoc. 1976;168:499-501.
24. Kawamura N, Shimada A, Morita T, et al. Intraperitoneal actino-
mycosis in a cat. Vet Rec. 2005;157:593-594.
25. Sharman MJ, Goh CS, Kuipers von Lande RG, et al. Intra-abdominal
actinomycetoma in a cat. J Feline Med Surg. 2009;11:701-705.
26. Frendin J, Greko C, Hellmén E, et al. Thoracic and abdominal wall
swellings in dogs caused by foreign bodies. J Small Anim Pract.
1994;35:499-508.
27. Johnson DE, Summers BA. Osteomyelitis of the lumbar verte-
bral in dogs caused by grass-seed foreign bodies. Aust Vet J.
1971;47:289-294.
28. Anvik JO, Lewis R. Actinomyces encephalitis associated with
hydrocephalus in a dog. Can Vet J. 1976;17:42-44.
29. Couto SS, Dickinson PJ, Jang S, et  al. Pyogranulomatous menin-
goencephalitis due to Actinomyces sp. in a dog. Vet Pathol.
2000;37:650-652.
30. Sturges BK, Dickinson PJ, Kortz GD, et al. Clinical signs, magnetic
resonance imaging features, and outcome after surgical and medi-
cal treatment of otogenic intracranial infection in 11 cats and 4
dogs. J Vet Intern Med. 2006;20:648-656.
31. Barrs VR, Nicoll RG, Churcher RK, et al. Intracranial empyema:
literature review and two novel cases in cats. J Small Anim Pract.
2007;48:449-454.
32. Stowater JL. Actinomycosis in the spinal canal of cat. Feline Pract.
1978;8:26-27.
33. Dhamborvorn T, Tritipsatit S, Meemongkoldilok S. Actinomycosis
of the urinary bladder. J Med Assoc Thai. 2001;84:109-112.
34. Ormsby AH, Bauer TW, Hall GS. Actinomycosis of the cholecystic
duct: case report and review. Pathology. 1998;30:65-67.
35. Harvey AM, Holt PE, Barr FJ, et  al. Treatment and long-term
follow-up of extrahepatic biliary obstruction with bilirubin choleli-
thiasis in a Somali cat with pyruvate kinase deficiency. J Feline Med
Surg. 2007;9:424-431.
36. Sykes JE, Kittleson MD, Pesavento PA, et al. Evaluation of the rela-
tionship between causative organisms and clinical characteristics of
infective endocarditis in dogs: 71 cases (1992-2005). J Am Vet Med
Assoc. 2006;228:1723-1734.
37. Mardis JS, Many Jr WJ. Endocarditis due to Actinomyces viscosus.
South Med J. 2001;94:240-243.
38. Cope VZ. Visceral actinomycosis. Ann R Coll Surg Engl.
1949;5:394-410.
39. Afolabi IR, Shashidhar VM. Carcinoma of the oesophagus mas-
querading as actinomycosis: a case report and a review of litera-
ture. Pac Health Dialog. 2004;11:94-95.
40. Batur Calis A, Ozbal AE, Basak T, et al. Laryngeal actinomycosis
accompanying laryngeal carcinoma: Report of two cases. Eur Arch
Otorhinolaryngol. 2006;263:783-785.
41. Frendin J. Diagnostic imaging of back pain. J Small Anim Pract.
1999;40:278-285.
42. Sivacolundhu RK, O’Hara AJ, Read RA. Thoracic actinomycosis
(arcanobacteriosis) or nocardiosis causing thoracic pyogranuloma
formation in three dogs. Aust Vet J. 2001;79:398-402.
43. Hoyles L, Falsen E, Pascual C, et al. Actinomyces catuli sp. nov.,
from dogs. Int J Syst Evol Microbiol. 2001;51:679-682.
44. Ng LS, Sim JH, Eng LC, et al. Comparison of phenotypic methods
and matrix-assisted laser desorption ionisation time-of-flight mass
spectrometry for the identification of aero-tolerant Actinomyces
spp. isolated from soft-tissue infections. Eur J Clin Microbiol Infect
Dis. 2011.
45. Fujita Y, Iikura M, Horio Y, et al. Pulmonary Actinomyces graeve-
nitzii infection presenting as organizing pneumonia diagnosed by
PCR analysis. J Med Microbiol. 2012;61:1156-1158.
46. Read RW, Zhang J, Albini T, et  al. Splendore-Hoeppli phenom-
enon in the conjunctiva: immunohistochemical analysis. Am J Ophthal-
mol. 2005;140:262-266.
47. Sykes JE, Weiss DJ, Buoen LC, et al. Idiopathic hypereosinophilic
syndrome in 3 Rottweilers. J Vet Intern Med. 2001;15:162-166.
48. Russo TA. Agents of actinomycosis. In: Mandell GL, Bennett JE,
Dolin R, eds. Principles and Practice of Infectious Diseases. 7th ed.
Philadelphia, PA: Elsevier; 2010:3209-3219.
49. Nelson JD, Hermann DW. Oral penicillin therapy for thoracic acti-
nomycosis. Pediatr Infect Dis. 1986;5:594-595.
50. Frendin J. Pyogranulomatous pleuritis with empyema in hunting
dogs. Zentralbl Vet Med. 1997;44:167-178.
51. Reiner SL, Harrelson JM, Miller SE, et al. Primary actinomycosis of an
extremity: a case report and review. Rev Infect Dis. 1987;9:581-589.
CHAPTER 43

Nocardiosis
Jane E. Sykes

spp. are ubiquitous soil saprophytes that degrade organic mat-

Overview of Nocardiosis
First Described: 1888, Alfort, France (Edmond Nocard)1
Causes: Nocardia spp. (order Actinomycetales, family
Nocardiaceae)
Geographic Distribution: Worldwide, although infections in
dogs and cats are most commonly reported from Brazil,
the western United States, and Australia
Mode of Transmission: Inhalation or cutaneous inoculation of
organisms in the environment, with opportunistic prolif-
eration in the face of impaired host immune defenses
Major Clinical Signs: Lethargy, weight loss, inappetence;
subcutaneous masses and nonhealing, crusted, or drain-
ing skin lesions; pulmonary nodules, masses and/or effu-
sion with cough and tachypnea; signs of dissemination
(neurologic signs, chorioretinitis, abdominal effusion or
masses, lameness)
Differential Diagnoses: Actinomycosis, streptomycosis,
mycobacteriosis, bartonellosis, fungal infections, neopla-
sia, leishmaniosis
Human Health Significance: Direct transmission of disease
from animals to humans does not occur, but humans have
developed disease after bites or scratches from healthy
dogs or cats.
Etiology and Epidemiology
ter. They are also found in fresh and salt water, in dust, and on
decaying plants and fecal matter.2 They can be carried mechani-
cally on the claws or skin. Infections are acquired via inhalation
of organisms or inoculation via puncture wounds. Transfer of
infection between animals does not occur.
Nocardiosis is much less frequently reported in dogs and cats
than actinomycosis, although nocardiosis is increasingly recog-
nized in both human medicine and companion animals in asso-
ciation with immunocompromise, such as occurs with potent
immunosuppressive drug therapy (especially cyclosporine).3-5
Where vaccination for canine distemper virus (CDV) is inad-
equate or not widely instituted, nocardiosis in dogs has been
associated with CDV-induced immunosuppression.6
The prevalence of nocardiosis and the predominant Nocar-
dia species that cause disease vary geographically. For example,
within the United States, human nocardiosis has been most
commonly reported from the southwestern states. Dry, dusty,
and windy conditions in these areas may facilitate aerosoliza-
tion and dispersal of nocardiae.7 Dogs and cats from Australia
and the western United States are most commonly infected with
Nocardia nova.8-10 Other species isolated from dogs and cats
are shown in Table 43-1.11-16 In humans, Nocardia brasiliensis
is the most common species isolated from patients that reside in
tropical locations such as the southwestern United States, Cen-
tral and South America, and Australia.
More than 60% of people with nocardiosis have underlying
immunosuppressive disorders such as AIDS, chronic obstructive
pulmonary disease, autoimmune diseases that require immuno-
suppressive drug therapy, solid organ transplantation, diabetes

Nocardia are filamentous, branching, gram-positive bacteria

that belong to the family Nocardiaceae (Figure 43-1). More
than 50 Nocardia species have been recognized since the advent
of molecular identification methods, approximately half of
which are human and animal pathogens.2 The advent of molec-
ular methods has also led to extensive revision of the taxonomy
of Nocardia spp. Species that belong to the former N. aster-
oides complex are now considered distinct species and include
Nocardia cyriacigeorgica (formerly N. asteroides sensu stricto),
Nocardia abscessus, Nocardia nova, Nocardia farcinica, and
Nocardia otitidiscaviarum.2 Knowledge of the species involved
is clinically important, because each Nocardia species differs
in its antimicrobial susceptibility patterns, epidemiology, and
pathogenicity. They also differ in their cell wall mycolic acid
content, which affects their ability to stain with acid-fast stains.
Like actinomycosis, nocardiosis is a suppurative to granu-
lomatous, localized, or disseminated opportunistic infection
caused by filamentous bacteria. However, unlike Actinomyces FIGURE 43-1  Gram-stained smear showing Nocardia spp. The organisms are
spp. (which are commensals of mucous membranes), Nocardia ­gram-positive, filamentous, branching, and slightly beaded.

409
410 SECTION 2  Bacterial Diseases

mellitus, and hemic neoplasia.17 Underlying immunosuppres-

TABLE 43-1 sive disease or drug treatment also seems to predispose dogs to
Nocardia Species Isolated from Dogs and Cats with Nocardiosis nocardiosis. Nocardiosis has been documented in several dogs
treated with cyclosporine and in a dog with lymphoma.3-5,9
Host Species Dogs Country Most affected cats have cutaneous infections that develop after
scratch or bite wounds.8,18 Accordingly, more than 75% to
Dogs N. nova United States, 80% of affected cats are male; in the author’s hospital, 9 of 10
Australia8,9 affected cats were male.8,9,19 Cats of any age can be affected,
N. paucivorans United States9 and no breed predilection has been identified.8 Some cats have
N. asiatica United States9 underlying disorders that predispose them to nocardiosis, such
N. abscessus United States5 as a history of renal transplantation, underlying retroviral infec-
N. otitidiscaviarum Brazil6 tion, or glucocorticoid administration. Others have no obvious
N. farcinica Australia8 underlying immunosuppressive disease.
Cats N. nova United States,
Australia9 Clinical Features
N. cyriacigeorgica Australia8
N. africana Japan,11 Brazil12 Signs and Their Pathogenesis
N. elegans Japan13 The pathogenicity of Nocardia spp. is influenced by the strain
N. brasiliensis United States14 and growth phase of the organism, and host susceptibility.
N. otitidiscaviarum Spain15 Virulent Nocardia strains are facultative intracellular patho-
N. tenerifensis United States16 gens that inhibit phagosome-lysosome fusion, neutralize phago-
somal acidification, resist oxidative burst, secrete superoxide
dismutase, and alter lysosomal enzymes within neutrophils and
macrophages.17 These effects are partly related to the content
and structure of mycolic acids within the bacterial cell wall,
which vary among strains and throughout the growth phase.
Filamentous log-phase cells in the environment can be highly
resistant to phagocytosis. Some strains have a greater propen-
sity to invade the central nervous system (CNS).19,20
The normal host response to infection is characterized by
an initial pyogenic inflammatory response, but a cell-mediated
immune response is necessary to destroy the organisms. Dimin-
ished host resistance, especially impaired cell-mediated immu-
nity (CMI), is a primary factor in susceptibility to nocardiosis
and the extent to which dissemination occurs.
Cutaneous-subcutaneous nocardiosis, the most common
form in cats (>75% of affected cats), is characterized by slow
and progressive circumferential spread of a nonhealing, drain-
A ing wound (Figure 43-2). Lesions are typically subacute to
chronic. Infections of feeding tube sites can also occur.21 Myce-
toma-like lesions in the inguinal area resemble those described
for rapidly growing mycobacterial infections (see Chapter 44),
with multiple draining sinuses. These may result from contami-
nation of “raking” injuries inflicted by the hindlimbs during
fighting behavior,8 or contamination of traumatic wounds from
penetrating plant material. Cutaneous-subcutaneous nocardio-
sis was documented in 8 of 9 dogs from Brazil6 but represents
the minority of canine nocardiosis cases in California and Aus-
tralia.8,9 Dogs with cutaneous involvement typically develop
draining wounds and masses, often on the head and limbs.
Osteomyelitis can occur in association with cutaneous lesions.
The pathogenesis of pulmonary or disseminated nocardiosis
is similar to that of the deep mycoses. Branching nocardial fila-
ments fragment into small, unicellular particles that are aero-
solized and inhaled, possibly in dust. Once within the lung,
B Nocardia spp. proliferate in the face of immune suppression,
which leads to formation of intrapulmonary masses or pneu-
FIGURE 43-2  Six-year-old male neutered domestic shorthair cat with cutaneous-
subcutaneous nocardiosis. The lesions progressed over at least a year after a cat bite monia, hilar lymphadenopathy, and/or extrapulmonary masses
abscess that was initially treated with amoxicillin-clavulanic acid. They consisted of nod- and pyothorax (Figure 43-3, A). Pulmonary nocardiosis is the
ular and crusted lesions on the head (A) and cervical region (B) that drained fluid and most common form of nocardiosis in humans and also occurs in
extended down the cervical region to the cranioventral thorax. The cat tested negative dogs and less commonly in cats. In dogs, it can have a peracute
for retroviruses. (Courtesy University of California, Davis, Veterinary Dermatology Service.) onset characterized by tachypnea, hemoptysis, hypothermia,

A B
FIGURE 43-3  Gross necropsy findings in a 4-year-old male neutered domestic shorthair cat with disseminated nocardiosis caused by Nocardia nova. The cat was a renal transplant
recipient. A, Multifocal to coalescing large, white to gray, caseous pulmonary masses are present within the lungs. B, The brain contained multifocal, white to gray foci that histologically
consisted of large numbers of neutrophils and filamentous bacteria. (Courtesy University of California, Davis Veterinary Anatomic Pathology Service.)
collapse, and death; however, subacute to chronic clinical signs
are more common.22-25 Co-infection with CDV is occasionally
reported.6,26,27 Disseminated nocardiosis occurs when organ-
isms in the lung erode into blood vessels and spread systemi-
cally, with abscess formation in a variety of organs. This results
in signs of lethargy, fever, inappetence, and signs that relate
to the location of the infectious process. Disseminated disease
occurs in cats as well as in dogs. The most frequently involved
extrathoracic organs after dissemination are skin and subcuta-
neous tissue, kidney, liver, spleen, lymph nodes, CNS, eye, bone,
and joints. In dogs and cats, CNS lesions can result in seizures
or neurologic signs that relate to the local effects of abscesses or
granulomas in the brain, meninges, or spinal cord (see Figure
43-3, B). CNS involvement is common in human nocardiosis.
Peritoneal nocardiosis has rarely been reported in cats, possibly
as a result of penetrating wounds to the abdomen.8,28
Physical Examination Findings
Physical examination findings in animals with cutaneous-
subcutaneous disease consist of chronic, sometimes crusted,
ulcerated, nonhealing draining wounds, and in cats may
involve the extremities, inguinal area, flank, head, bridge of
the nose, and neck (see Figure 43-2). Animals with pulmonary
or systemic involvement may have a thin body condition, leth-
argy, and fever. Tachypnea and cough may be present, and
lung sounds may be increased (with bronchopneumonia) or
decreased (with pyothorax). Involvement of the liver, spleen,
and lymph nodes may result in hepatomegaly, splenomegaly,
and/or peripheral or abdominal lymphadenomegaly. Oph-
thalmic examination may reveal chorioretinitis. Bone or joint
infection results in focal limb swelling and lameness. Animals
with CNS involvement may show mental obtundation, aniso-
coria, decreased menace and pupillary light responses, head
tilt, nystagmus, decreased gag reflexes, and/or delayed or
absent placing reactions.
Diagnosis
A diagnosis of nocardiosis is usually suspected based on the
presence of persistent pyogenic to pyogranulomatous inflamma-
tory lesions and the presence of filamentous bacteria, together
CHAPTER 43  Nocardiosis 411
with a history of immunosuppression, although the last is not
always present. Confirmation of the diagnosis requires culture.
Laboratory Abnormalities
Complete Blood Count and Serum Biochemical Tests
Hematologic abnormalities in nocardiosis are similar to those
with actinomycosis (nonregenerative anemia, neutrophilic leu-
kocytosis with a left shift, monocytosis, and sometimes marked
hyperglobulinemia). However, in immunosuppressed animals,
lymphopenia, monocytopenia, and eosinopenia may be pres-
ent. Hypercalcemia associated with granulomatous disease was
reported in a cat with nocardiosis.29
Cytologic Examination of Body Fluids
Examination of pleural effusion, bronchoalveolar lavage fluid,
and aspirates of abscesses from animals with nocardiosis typi-
cally reveals suppurative to pyogranulomatous inflammation,
with large numbers of degenerate neutrophils. Gram-positive,
often partially or weakly acid-fast, beaded, filamentous organ-
isms that branch at right angles may be observed individually
or in loose aggregates (see Figure 43-1). The filaments may also
fragment to form rods and coccoid forms. In contrast, Acti-
nomyces spp. is not acid fast, and Mycobacterium spp. do not
branch. When the infecting Nocardia sp. is not acid fast, it may
not be distinguishable from an Actinomyces sp. organism. With
Romanowsky-type stains, organisms are eosinophilic or baso-
philic and have a beaded appearance. They are usually around
0.5 µm in diameter and up to 30 µm long, but thicker organ-
isms were described in a dog infected with N. abscessus (2 µm).5
Macroaggregates (i.e., sulfur granules) occur infrequently in
effusions. In contrast to actinomycosis, mixed bacterial popula-
tions in deep tissue sites are rarely present.
Diagnostic Imaging
Plain Radiography
Radiographs of cutaneous-subcutaneous lesions can reveal
soft tissue swelling with or without associated bone lysis and
periosteal proliferation. The radiographic appearance of pul-
monary lesions varies and includes multiple, diffuse pulmonary
nodules; intrapulmonary or extrapulmonary solitary masses;
focal or diffuse bronchointerstitial to alveolar infiltrates; lobar
412 SECTION 2  Bacterial Diseases

consolidations; pleural effusions; and, often, dramatic hilar Microbiologic Tests

lymphadenopathy (Figure 43-4).
Sonographic Findings
Abdominal ultrasound findings in dogs and cats with dissemi-
nated nocardiosis have not been described in detail because dis-
seminated disease with abdominal organ involvement is rare.
However, intraparenchymal mass lesions and flocculent ascites
fluid might be expected.
Advanced Imaging
Nodular lung lesions similar to those described in humans were
identified with computed tomography in one dog with dissemi-
nated nocardiosis.5 Computed tomographic and MRI abnor-
malities in dogs and cats with confirmed CNS nocardiosis have
not been reported. One dog with suspected nocardiosis based
on histopathology had a large, T2-hyperintense circumscribed
lesion in the occipital cortex that was consistent with a brain
abscess, together with widespread cerebral edema and hernia-
tion of the cerebellum through the foramen magnum.4
FIGURE 43-4  Lateral thoracic radiograph of a 4-year-old male neutered domestic
shorthair cat with disseminated nocardiosis. Pleural effusion is present and obscures the
cardiac silhouette. A mass is associated with the caudal aspect of the left caudal lung
lobe.
Diagnostic assays available for Nocardia infection in dogs and
cats are shown in Table 43-2.
Isolation and Identification
Nocardia spp. grow aerobically at a wide temperature range on
simple media (e.g., Sabouraud’s glucose agar, blood agar). Organ-
isms are usually recovered in pure cultures, and colonies are often
visible after 2 days. However, in some cases, 2 to 4 weeks of
incubation may be necessary, and the organism may be over-
grown by contaminating bacteria. Thus, the laboratory should
be alerted if nocardiosis is suspected, so that selective media and
long incubation times are used. Colonies are smooth and moist,
or rugose with a powdery surface due to aerial filamentation, and
may be pigmented. Organisms may then be identified based on
their microscopic appearance and their ability to take up acid fast
stains. However, not all pathogenic strains of Nocardia species
are acid fast.19,20,30 L-form Nocardia spp., which are cell wall–
deficient variants, have been associated with disease in people
and a dog.31 These bacteria require special media for isolation
and culture, similar to mycoplasmas (see Chapter 39).
Traditionally, Nocardia species have been distinguished based
on their growth characteristics and antibiotic susceptibility pat-
terns; however, molecular methods provide a more reliable and
rapid means of speciation. Sequence analysis of PCR products
from the 16S rRNA gene identifies most pathogenic Nocardia
spp., but differentiation of closely related species may require
analysis of other genes, multilocus sequence typing, or DNA-
DNA hybridization.2,17 DNA-DNA hybridization is considered
the gold standard for nocardial species determination.2 Matrix-
assisted laser desorption/ionization–time of flight (MALDI-
TOF) mass spectrometry shows promise for rapid and reliable
identification of Nocardia species in clinical microbiology lab-
oratories.32 PCR assays have also been used to directly detect
Nocardia spp. in clinical specimens, without prior isolation.2
Because Nocardia organisms are ubiquitous in soil and may
be inhaled by healthy animals, the isolation of small numbers
of organisms from ulcerated skin lesions or the respiratory tract
may not be clinically significant and must be interpreted in con-
junction with clinical signs and history of immune compromise.
Repeated positive cultures, pure cultures, or isolation of large

TABLE 43-2
Diagnostic Assays Available for Nocardia Infection in Dogs and Cats
Assay Specimen Type Target Performance
Culture Aspirates of purulent material Nocardia spp. Grows on most simple media. May require
(e.g., abscesses, pleural effusion); several weeks’ incubation. Because Nocardia
whole blood; tissue specimens spp. are soil saprophytes, isolation from skin
obtained by biopsy or at nec- lesions does not imply disease causation.
ropsy (e.g., subcutaneous tissue, PCR and sequencing may be required to
lung tissue) identify the species present.
Cytology or histo- Aspirates or tissue specimens Nocardia spp. Organisms are gram-positive, variably acid-
pathology with organisms fast, and are not typically accompanied
organism detection by sulfur granules, other bacteria, or the
using special stains Splendore-Hoeppli phenomenon. Definitive
diagnosis requires culture because non–acid-
fast Nocardia can resemble Actinomyces spp.
 CHAPTER 43  Nocardiosis 413

numbers of the organism also suggest nocardiosis, and isola-
tion of a single colony from a normally sterile site is significant.
Visualization of gram-positive filamentous bacteria in cytology
specimens in association with acute inflammatory cells also
assists in determination of clinical significance.
The susceptibility of Nocardia spp. to antimicrobial drugs
varies considerably between different Nocardia species, and so
species identification can be used to guide antimicrobial drug
selection (see Treatment, later) (Table 43-3). Susceptibility test-
ing for Nocardia spp. is difficult and should be done by labora-
tories with special expertise. Susceptibility testing may be helpful
when there is a failure to respond to therapy or relapse occurs
from drug resistance. It may also be useful if there are concerns
that relate to adverse drug reactions to sulfonamides, the initial
treatment of choice (e.g., in dogs with immune-mediated disease
that is complicated by cyclosporine-associated nocardiosis).
Testing is also indicated when N. farcinica is present, which
tends to be highly resistant to antimicrobial drugs, or when a
nocardial species with unpredictable susceptibility is isolated.
Pathologic Findings
Nocardiosis is characterized by suppurative necrosis and abscess
formation and infrequently granulomas (see Figure 43-3). Gross
lesions in organs such as the spleen, lung, liver, and the brain
typically consist of numerous small (1 mm) to large (several
centimeters), discrete or coalescing, intraparenchymal white or
gray-white nodules. On cut section, nodules appear caseous to
purulent. Lymph nodes are enlarged, often massively, and are
firm to fluctuant with a caseous to purulent core. A reddish-
brown exudate may be present in the pleural or peritoneal space
or within abscesses. Although formation of sulfur granules by
Nocardia spp. is uncommon compared with Actinomyces spp.,
small granules are occasionally found in skin lesions and pleural
and peritoneal fluid.10,22,28,33
Histopathology usually reveals a central region of necro-
sis and suppuration, which, depending on the host immune
response, may be surrounded by macrophages, lymphocytes,
and plasma cells (Figure 43-5, A). In chronic cutaneous-
subcutaneous infections, pyogranulomatous foci may be inter-
spersed within dense fibrous tissue. Plant material may be
observed if plants were involved in inoculation of Nocardia
spp. into tissues, but this is more common with actinomycosis.
Nocardia filaments are usually present in abundance within
regions of necrosis and suppuration. They are poorly visible
in tissue sections stained with hematoxylin and eosin or with
Gridley’s fungal or periodic acid–Schiff. Organisms can be
stained with Gram stain (see Figure 43-5, B) or methenamine
silver preparations,5 especially with prolonged silver nitrate
exposure. They are usually partially acid fast and have the same
appearance described previously in the Cytology section. In
chronic skin infections, tissue granules characterized by colo-
nies arranged in large, rosette-like arrays have been described.
In contrast to actinomycosis, other bacteria and the Splendore-
Hoeppli phenomenon are not generally present.
Treatment and Prognosis
Treatment
Successful treatment of nocardiosis relies on the combina-
tion of appropriate antimicrobial therapy combined with the

TABLE 43-3
Suggested Antimicrobial Drug Selection for Treatment of Pathogenic Nocardia spp. Based on Minimum Inhibitory
Concentration Data
Appropriate Antimicrobial
Nocardia Species Drug Choices Antimicrobial Drugs to Avoid
N. abscessus Ampicillin, amoxicillin–clavulanic acid, Imipenem, ciprofloxacin, clarithromycin
ceftriaxone, linezolid, amikacin and
gentamicin, sulfamethoxazole
N. brevicatena/paucivorans complex Ampicillin, amoxicillin–clavulanic acid, Gentamicin, clarithromycin
ceftriaxone, amikacin, sulfamethoxazole,
ciprofloxacin
N. nova complex (includes N. nova, Ampicillin, sulfamethoxazole, Amoxicillin–clavulanic acid
N. africana) ­erythromycin, clarithromycin,
­ceftriaxone, imipenem, amikacin
N. farcinica Amikacin, sulfamethoxazole, ciprofloxacin Ampicillin, broad-spectrum cephalospo-
(most isolates), imipenem (most isolates) rins, clarithromycin, aminoglycosides
other than amikacin
N. cyriacigeorgica Ceftriaxone, amikacin, imipenem Ampicillin, amoxicillin–clavulanic acid,
clarithromycin, ciprofloxacin
N. brasiliensis Minocycline, amoxicillin–clavulanic acid, Ampicillin, ciprofloxacin, clarithromycin
sulfamethoxazole
N. otitidiscaviarum Gentamicin, amikacin, sulfamethoxazole, All β-lactam antibiotics
ciprofloxacin

Data from Brown-Elliott BA, Brown JM, Conville PS, et al. Clinical and laboratory features of the Nocardia spp. based on current molecular tax-
onomy. Clin Microbiol Rev 2006;19:259-282.
414 SECTION 2  Bacterial Diseases
use of surgical drainage or debridement. Chronic, extensive
lesions may require surgical debridement or resection. Appro-
priate antimicrobial therapy that includes surgical drainage is
not always effective, possibly because of an inadequate host
immune response.8 Existing immunosuppressive drug treatment
should be discontinued or reduced, provided it does not cause
relapse of a life-threatening underlying disease process.
The initial selection of an antimicrobial drug or drug combina-
tion should take into account the site of the infection, the host
immune status, the infecting Nocardia species (see Table 43-3),
A nocardiosis in human patients.2 A combination of amikacin and
B Prognosis
FIGURE 43-5  Histopathologic findings in nocardiosis. A, Severe, necrotizing
pyogranulomatous inflammation is present with intralesional bacteria, which are barely
visible. H&E stain. B, Gram staining revealed tangles of filamentous, branching gram-
positive bacteria. Brown and Benn stain.
TABLE 43-4
Medications That Could Be Used to Treat Dogs and Cats with Nocardiosis *
Drug Dose (mg/kg)
Trimethoprim-sulfamethoxazole 30
Amikacin† 10-14 (cat), 15-30 (dog)
Imipenem-­cilastatin† 2-5
*Pending species identification with or without susceptibility testing.
†Amikacin and imipenem-cilastatin are generally used in combination with each other or with trimethoprim-sulfamethoxazole.
and, if available, susceptibility test results.17 However, in  vitro
susceptibilities do not always translate into a clinical response
in vivo. Sulfonamides, including trimethoprim-sulfonamide com-
binations (TMS), are the first line antimicrobial drugs for treat-
ment of nocardiosis (Table 43-4). Treatment durations from
1 to 3 months are recommended in people with cutaneous infec-
tions, up to 6 months for uncomplicated pulmonary infections,
and 12 months or longer for systemic infections or infections in
those who are immunocompromised.17 Clinical improvement is
generally observed within 7 to 10 days of starting treatment. High
doses of trimethoprim-sulfas given for long periods to dogs (and
to a lesser extent, cats) can produce a variety of adverse effects
such as keratoconjunctivitis sicca and myelosuppression (see
Chapter 8). If adverse drug reactions occur, selection of drugs
other than TMS should ideally be based on susceptibility test
results. If these are not available, choices should be made based
on published susceptibility data for different Nocardia species (see
Table 43-3). Nocardia nova is susceptible to amoxicillin but
resistant to amoxicillin-clavulanic acid because of induction of
chromosomal β-lactamase by clavulanic acid.8,10 Substitution of
antimicrobial drugs within a drug class may not provide effec-
tive treatment; for example, although susceptible to amikacin,
N. farcinia isolates are resistant to gentamicin, and although sus-
ceptible to minocycline, N. brasiliensis isolates are mostly resis-
tant to doxycycline.34 Two- or three-drug combinations including
TMS, amikacin, and either ceftriaxone or imipenem have been
used, pending the results of susceptibility tests to treat severe
imipenem should be effective against all isolates. Combination
treatment with amikacin and TMS was suggested for cats with
N. farcinica infections, which generally require an aggressive,
combination drug treatment.8 After susceptibility testing, mono-
therapy with linezolid has consistently been used with success to
treat some refractory Nocardia infections in humans.35 Nocardia
isolates from dogs and cats have high minimum inhibitory con-
centrations for fluoroquinolones,36 which are not generally recom-
mended for treatment.
If CNS disease is present, either high doses of TMS given
parenterally, or drugs with excellent CNS penetration, which
include third-generation cephalosporins, carbapenems, or line-
zolid should be considered. The use of linezolid in veterinary
patients is controversial because it is an important drug for
resistant bacterial infections in humans (see Chapter 8).
In a review of 53 dogs with nocardiosis, 50% of the dogs died
and 39% were euthanized.24 Of 36 cats with nocardiosis, 16 were
either euthanized or died.8 The high mortality rate may relate to
underlying immunosuppressive disease or immunosuppressive
Route Interval (hours)
PO, IV 12
IV, IM, SC 24
IV 8

drug treatment, delayed diagnosis, and inappropriate treatment.
With earlier diagnosis and more aggressive, multidrug therapy, mor-
tality of nocardiosis in animals may decrease to the rate reported in
people. Only 20% of humans with primary infections died, whereas
42% of patients with predisposing conditions and more than 50%
of patients with systemic or CNS nocardiosis died.19
Immunity and Vaccination
Immunity to nocardiosis is dependent on intact CMI. There are
no vaccines for nocardiosis.
Prevention
Prevention of nocardiosis in cats involves housing cats indoors
and limiting their fighting or biting activity. Any wounds should
CASE EXAMPLE
Signalment: “Bob,” a 6-year-old male neutered domestic
shorthair cat from San Francisco, CA
History: Bob, a renal transplant recipient, was evaluated
for a 2-week history of decreased activity and progressive
inappetence and lethargy. He was taken to a local
emergency clinic where blood work showed only mild
hyponatremia (130 mmol/L) and a white cell count of 19,000
cells/µL. No abnormalities were detected on abdominal
ultrasound examination. Bob was treated overnight with
IV fluids and sent home with instructions to administer
amoxicillin-clavulanic acid (12.5 mg/kg PO q12h). However,
the cat’s condition failed to improve, and so he was taken
to a local veterinary clinic 3 days later. Further laboratory
testing showed hyponatremia, hypoalbuminemia, and mild
hyperbilirubinemia. Urinalysis showed a specific gravity of
1.054, 3+ bilirubin, 1+ hemoprotein, 0-2 WBC/HPF, 6-10 RBC/
HPF, and no bacteria or casts. Aerobic bacterial urine culture
was negative. The following day Bob became tachypneic
and pleural effusion was identified. In-house examination
of the effusion fluid revealed cloudy fluid that contained
large numbers of erythrocytes, degenerate neutrophils,
and macrophages. No bacteria were seen with Gram stain.
Bob was then referred to the University of California, Davis,
Veterinary Medical Teaching Hospital for further evaluation.
Bob had received a renal transplant at 3.5 years of age and
since then had been treated with prednisone (0.5 mg/kg PO
q12h) and cyclosporine (4 mg/kg PO q12h). A recent whole
blood trough cyclosporine concentration was 1100 ng/mL.
Physical Examination:
Body Weight: 5.1 kg
General: Severely obtunded, T = 100.8°F (38.2°C), HR = 240
beats/min, RR = 55 breaths/min, mild generalized icterus,
mucous membranes pink to icteric, CRT = 1 s, hydrated.
Eyes, Ears, Nose, and Throat: The only clinically significant
abnormalities related to the eyes. Anisocoria was present
with the left pupil larger than the right. Direct and
consensual pupillary light reflexes were present, and the
CHAPTER 43  Nocardiosis 415
be carefully examined for foreign material and cleaned promptly.
Disease in both dogs and cats may be prevented by avoidance
of excessive immunosuppression with potent drugs such as
cyclosporine.
Public Health Aspects
No cases of human nocardiosis acquired from direct con-
tact with an animal have been reported; however, several
cases of cutaneous nocardiosis transmitted to people by cat
scratches have been documented.37-40 Humans with impaired
CMI (such as those receiving immunosuppressive drug ther-
apy or those with HIV infection) should wear gloves and
practice hand washing after handling pets with nocardiosis,
or another individual in the household should treat these
animals.
cornea and anterior chamber were clear with no ocular
discharge. Fundoscopic examination showed focal areas of
tapetal hyperreflectivity and focal dark and dull lesions with
peripheral hyperreflectivity.
Musculoskeletal: BCS 7/9, with generalized weakness,
recumbency and unwillingness/inability to walk.
Respiratory: Tachypnea with a shallow respiratory pattern was
identified. Decreased lung sounds were present ventrally.
All Other Systems: No clinically significant abnormalities were
present. The transplanted kidney could be palpated in the
right abdomen. The urinary bladder was small.
Neurologic Examination:
Mentation: Obtunded.
Gait/Posture: The cat had a tendency to lean to the right, but
could not or would not walk more than a few steps.
Cranial Nerves: Menace responses were absent bilaterally.
Postural Reactions: Delayed placing reactions were identified
in all four limbs.
Spinal Reflexes and Panniculus Reflex: Normal.
Neuroanatomic Location: Cerebrothalamic, possibly right
sided.
Laboratory Findings:
CBC:
HCT 30.4% (30%-50%), MCV 46.5 fL (42-53 fL)
MCHC 32.6 g/dL (30-33.5 g/dL)
Reticulocytes 18,000 cells/µL (7000-60,000 cells/µL)
WBC 22,860 cells/µL (4500-14,000 cells/µL)
Neutrophils 19,660 cells/µL (2000-9000 cells/µL)
Band neutrophils 2286 cells/µL
Lymphocytes 457 cells/µL (1000-7000 cells/µL)
Monocytes 229 cells/µL (50-600 cells/µL)
Eosinophils 229 cells/µL (150-1100 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL).
Platelets clumped but adequate. Neutrophils had slight toxic
change, and band neutrophils had marked toxic change.
Serum Chemistry Profile:
Sodium 136 mmol/L (151-158 mmol/L)
Potassium 3.5 mmol/L (3.6-4.9 mmol/L)
Chloride 98 mmol/L (113-121 mmol/L)
Bicarbonate 18 mmol/L (15-21 mmol/L)
Continued
416 SECTION 2  Bacterial Diseases
Phosphorus 4.3 mg/dL (3.2-6.3 mg/dL)
Calcium 8.3 mg/dL (9.4-11.4 mg/dl)
BUN 48 mg/dL (18-33 mg/dL)
Creatinine 0.8 mg/dL (1.1-2.2 mg/dL)
Glucose 140 mg/dL (73-134 mg/dL)
Total protein 4.8 g/dL (6.6-8.4 g/dL)
Albumin 1.4 g/dL (1.9-3.9 g/dL)
Globulin 3.4 g/dL (2.9-5.3 g/dL)
ALT 19 U/L (28-106 U/L)
AST 27 U/L (12-46 U/L)
ALP 5 U/L (14-71 U/L)
Gamma GT 0 U/L (0-4 U/L)
Cholesterol 141 mg/dL (89-258 mg/dL)
Total bilirubin 1.5 mg/dL (0-0.2 mg/dL).
Imaging Findings:
Thoracic Radiographs: See Figure 43-4. There was a moderate
amount of pleural effusion. A mass was associated with
the caudal aspect of the left caudal lung lobe. The cardiac
silhouette was completely obscured on the lateral projection.
On the ventrodorsal projection, there appeared to be a mass
effect superimposed over the caudal border of the cardiac
silhouette that deformed the left and right caudal lung lobes.
Abdominal Ultrasound: The native kidneys were markedly
diminished in size, were irregular in contour, and
had a markedly irregular parenchyma with a lack of
corticomedullary distinction. The transplanted kidney
appeared within normal limits. The muscularis layer of the
small intestine was mildly thickened diffusely. A Y-shaped
region of mineralization was identified within the right liver
lobes, which was likely associated with the biliary system.
Treatment and Outcome: Thoracocentesis was performed,
and 35 mL of turbid red fluid was removed. Unfortunately,
the cat became progressively more agitated and tachypneic,
thoracocentesis was aborted, and Bob was placed in a
cage with oxygen supplementation. Preliminary cytologic
examination of the fluid yielded results that were consistent
with referring veterinarian’s findings. Treatment with IV
crystalloid fluids (0.9% NaCl with 20 mEq/L KCl at 12 mL/
hr), enrofloxacin (2.5 mg/kg IV q12h), and ampicillin (22 mg/
kg IV q8h) was initiated, and pleural fluid was submitted to
the laboratory for cytologic examination and aerobic and
anaerobic bacterial culture. The plan was to stabilize Bob
with this treatment until additional thoracocentesis and
chest tube placement could be performed. Differential
diagnoses considered were cryptococcosis, toxoplasmosis,
SUGGESTED READINGS
Brown-Elliott BA, Brown JM, Conville PS, et al. Clinical and laboratory
features of the Nocardia spp. based on current molecular taxonomy.
Clin Microbiol Rev. 2006;19:259-282.
MacNeill AL, Steeil JC, Dossin O, et al. Disseminated nocardiosis caused
by Nocardia abscessus in a dog. Vet Clin Pathol. 2010;39:381-385.
Malik R, Krockenberger MB, O’Brien CR, et al. Nocardia infections in
cats: a retrospective multi-institutional study of 17 cases. Aust Vet J.
2006;84:235-245.
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nue sous le nom de farcin. Ann Inst Pasteur (Paris). 1888;2:293-302.
FIP, a systemic bacterial infection, or neoplasia. However,
10 hours after admission, Bob developed respiratory and
cardiac arrest. The owner elected euthanasia.
Necropsy Findings: Gross necropsy findings consisted of 120
mL of flocculent, serosanguinous fluid within the thoracic
cavity and multifocal to coalescing, variably sized, white to
gray, firm to caseous nodules that largely effaced the caudal
lung lobes and extended through the pleural surface, with
adhesions to the thoracic diaphragm and pleura (see Figure
43-3, A). A 3-mm nodule was also present in the wall of
the left ventricle. The peritoneal cavity contained multiple
adhesions from the small intestines to the omentum and
the stomach to the left abdominal wall. The right medial
liver lobe contained a focal, white to gray mass that was 10
cm in diameter and oozed purulent material when cut. The
spleen contained multiple, white, raised, pinpoint foci. The
native kidneys were shrunken, pale, irregular, and had loss of
corticomedullary definition. The right transplanted kidney
contained 3-4, tan, firm, homogenous cortical nodules that
ranged from 2 to 4 mm in diameter. On cut section, the brain
contained multifocal, randomly distributed, soft, white to
gray, homogenous foci that measured 1-4 mm in diameter
(see Figure 43-3, B). Histopathology of the lung, pleural,
myocardium, thoracic diaphragm, spleen, liver, transplanted
kidney, adrenal medulla, stomach, salivary gland, brain,
and meninges revealed chronic, severe, necrotizing
pyogranulomatous inflammation with intralesional
branching filamentous bacteria. There was also chronic,
moderate, multifocal and necrotizing pyogranulomatous
choroiditis and cyclitis in both eyes. The bacteria were gram
positive but were not acid fast (see Figure 43-5, B).
Microbiologic Testing: Aerobic and anaerobic bacterial
culture (liver, lung specimens collected at necropsy): Small
numbers of Nocardia nova. No other bacteria were cultured.
Diagnosis: Disseminated Nocardia nova infection
Comments: In this case, nocardiosis followed a rapid
clinical course, as can occur in human patients with severe
immunodeficiency. The trough whole blood cyclosporine
concentration was high; the goal of treatment is to obtain
blood concentrations of approximately 300 to 500 ng/
mL, although the optimum concentration that achieves
adequate but not excessive immunosuppression in cats is
not known.18 Treatment with antimicrobial drugs was not
effective given the advanced nature and severity of the
disease in this cat.
2. Brown-Elliott BA, Brown JM, Conville PS, et al. Clinical and labo-
ratory features of the Nocardia spp. based on current molecular
taxonomy. Clin Microbiol Rev. 2006;19:259-282.
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infection in a dog treated with cyclosporin and ketoconazole. N Z
Vet J. 2010;58:265-268.
4. Smith PM, Haughland SP, Jeffery ND. Brain abscess in a dog
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diosis caused by Nocardia abscessus in a dog. Vet Clin Pathol.
2010;39:381-385.
6. Ribeiro MG, Salerno T, Mattos-Guaraldi AL, et al. Nocardiosis: an
overview and additional report of 28 cases in cattle and dogs. Rev
Inst Med Trop Sao Paulo. 2008;50:177-185.

7. Saubolle MA, Sussland D. Nocardiosis: review of clinical and labo-
ratory experience. J Clin Microbiol. 2003;41:4497-4501.
8. Malik R, Krockenberger MB, O’Brien CR, et  al. Nocardia infec-
tions in cats: a retrospective multi-institutional study of 17 cases.
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9. Sykes JE. Unpublished observations. 2012.
10. Hirsh DC, Jang SS. Antimicrobial susceptibility of Nocardia nova
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11. Hattori Y, Kano R, Kunitani Y, et al. Nocardia africana isolated
from a feline mycetoma. J Clin Microbiol. 2003;41:908-910.
12. de Farias MR, Werner J, Ribeiro MG, et al. Uncommon mandibu-
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13. Harada H, Endo Y, Sekiguchi M, et al. Cutaneous nocardiosis in a
cat. J Vet Med Sci. 2009;71:785-787.
14. Ajello L, Walker WW, Dungworth DL, et al. Isolation of Nocar-
dia brasiliensis from a cat with a review of its prevalence and geo-
graphic distribution. J Am Vet Med Assoc. 1961;138:370-376.
15. Ramos-Vara JA, Wu CC, Lin TL, et  al. Nocardia tenerifensis
genome identification in a cutaneous granuloma from a cat. J Vet
Diagn Invest. 2007;19:577-580.
16. Luque I, Astorga R, Tarradas C, et al. Nocardia otitidiscaviarum
infection in a cat. Vet Rec. 2002;151:488.
17. Sorrell TC, Mitchell DH, Iredell JR, et  al. Nocardia species. In:
Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of
Infectious Diseases. Philadelphia, PA: Elsevier; 2010:3199-3207.
18. Edwards DF. Actinomycosis and nocardiosis. In: Greene CE, ed.
Infectious Diseases of the Dog and Cat. 3rd ed. St Louis, MO:
Saunders Elsevier; 2006:451-461.
19. Beaman BL, Beaman L. Nocardia species: host-parasite relation-
ships. Clin Microbiol Rev. 1994;7:213-264.
20. McNeil MM, Brown JM. The medically important aerobic acti-
nomycetes: epidemiology and microbiology. Clin Microbiol Rev.
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21. Kadar E, Sykes JE, Kass PH, et  al. Evaluation of the prevalence
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22. Campbell B, Scott DW. Successful management of nocardial
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23. Cross RF, Nagao WT, Morrison RH. Canine nocardiosis; a report
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28. Tilgner SL, Anstey SI. Nocardial peritonitis in a cat. Aust Vet J.
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29. Mealey KL, Willard MD, Nagode LA, et al. Hypercalcemia associ-
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30. Lerner PI. Nocardiosis. Clin Infect Dis. 1996;22:891-903; quiz
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31. Buchanan AM, Beaman BL, Pedersen NC, et al. Nocardia asteroi-
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idiopathic polyarthritis. J Clin Microbiol. 1983;18:702-708.
32. Verroken A, Janssens M, Berhin C, et  al. Evaluation of matrix-
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36. Govendir M, Norris JM, Hansen T, et al. Susceptibility of rapidly
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37. Astudillo L, Dahan S, Escourrou G, et al. Cat scratch responsible
for primary cutaneous Nocardia asteroides in an immunocompetent
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CHAPTER 44
Mycobacterial Infections
Jane E. Sykes and Danièlle A. Gunn-Moore
Overview of Mycobacterial Infections in Dogs
and Cats
First Described: Evidence of tuberculosis in humans dates
back to 2400 to 3400 bc, but the causative agent was not
demonstrated until 1882 (Robert Koch).1
Causes: Mycobacterium spp. (order Actinomycetales, family
Mycobacteriaceae)
Geographic Distribution: Worldwide, but the distribution of
different mycobacterial species varies.
Major Clinical Signs: Cutaneous nodular, ulcerated, or drain-
ing skin lesions; peripheral or internal lymphadenopathy;
pneumonia with cough and/or tachypnea; osteomyelitis;
granulomatous or pyogranulomatous infiltrates in a vari-
ety of abdominal organs and, rarely, the central nervous
system and eye.
Differential Diagnoses: Neoplasia (especially lymphoma),
feline infectious peritonitis (cats), tularemia, nocardiosis,
actinomycosis, rhodococcosis, bartonellosis, leishmani-
asis, and fungal infections
Human Health Significance: Transmission of mycobacteria
from dogs or cats to humans has not been reported. How-
ever, the potential for transmission of M. tuberculosis and
M. bovis to humans exists. Humans acquire infection by
other mycobacteria from environmental sources.
M
ycobacterium spp. are aerobic, nonmotile, non–spore-
forming, gram-positive, acid-fast pleomorphic bacilli
that cause chronic infections in humans and animals.
Mycobacterium spp. have a high cell wall mycolic acid content,
which causes them to retain a pink color when stained with
acid-fast stains such as Ziehl-Neelsen or Kinyoun stains and
examined under the microscope. As a result, the term “acid-fast
bacilli” (AFB) is often associated with mycobacteria, although
Nocardia is variably acid fast (see Chapter 43).
Mycobacterium spp. belong to the family Mycobacteriaceae
and are grouped into those that belong to the Mycobacterium
tuberculosis complex (MTBC), the Mycobacterium avium com-
plex (MAC), the lepromatous mycobacteria, and nontubercu-
lous mycobacteria other than M. avium (Table 44-1). Disease
caused by these groups of microorganisms differs in its epide-
miology, pathogenesis, clinical presentation, zoonotic potential,
and response to antimicrobial treatment. Accordingly, each
group is considered separately in this chapter.
418
Mycobacterium tuberculosis Complex
Etiology and Epidemiology
MTBC bacteria include the slowly growing, host-associated
bacteria Mycobacterium tuberculosis, Mycobacterium bovis,
Mycobacterium microti, Mycobacterium africanum, Myco-
bacterium pinnipedii, Mycobacterium caprae, and Mycobac-
terium canettii. These organisms cause tuberculosis in humans
and animals. Only M. tuberculosis, M. bovis, and M. microti
infect dogs and cats (Table 44-2). M. africanum and M. canettii
are rare causes of human tuberculosis in Africa, M. pinnipedii
infects seals, and M. caprae is primarily a ruminant pathogen.
MTBC organisms share greater than 95% DNA-DNA homol-
ogy, and so are difficult to differentiate from one another, even
with many molecular methods. Unlike other mycobacterial spe-
cies, MTBC bacteria do not survive well in the environment,
although M. bovis can survive months in feces or carcasses.
Humans are the only reservoir host for M. tuberculosis, which
is responsible for more than 90% of tuberculosis in humans.
Cats are resistant to infection with M. tuberculosis. Disease due
to M. tuberculosis has been reported in dogs that reside in vari-
ous parts of the United States, Africa, France, and Switzerland.2-9
Affected dogs from Europe have had travel histories; one was
from western Africa and the other was a stray from southern
Europe.2,3 Dogs are usually infected after prolonged aerosol
exposure to contaminated human respiratory secretions, so the
disease is a “reverse zoonosis” (or anthropozoonosis). Thus,
disease occurs in dogs where tuberculosis occurs in humans. In
the United States, M. tuberculosis infection is most prevalent in
humans from the northeast/mid-Atlantic states, southern states,
Alaska, California, and Nevada (Figure 44-1). Dogs have not
transmitted M. tuberculosis infection to humans despite pro-
longed close contact in some situations,7 possibly because the
pulmonary cavitary lesions that are required for transmission
do not develop to the same extent in dogs as they do in humans.
However, organisms have been detected in dogs’ sputum, so
transmission remains possible.
Cats are most often infected with M. microti or M. bovis.10
Rodents (especially voles and shrews) are reservoir hosts for
M. microti, and cattle and a variety of wildlife species are res-
ervoir hosts for M. bovis. The overwhelming majority of feline
infections with M. microti and M. bovis have been reported from
the United Kingdom, where M. microti infection is endemic in
voles, wood mice, and shrews, and M. bovis infection is endemic
in Eurasian badgers and cattle. Infections with M. microti and
M. bovis comprise a third of feline infections with culturable
mycobacteria in the United Kingdom.10 Cats infected with
M. bovis tend to be young adult cats (median age 3 years),
 CHAPTER 44  Mycobacterial Infections 419

TABLE 44-1
Mycobacterial Pathogens of Clinical Significance in Dogs and Cats
Group Mycobacterial Species Description
M. tuberculosis complex M. tuberculosis, M. bovis, M. microti Host-associated, slow-growing species. Cause cutaneous
and disseminated granulomatous disease in dogs (all three
species) and cats (M. bovis and M. microti)
M. avium-intracellulare M. avium subsp. avium, M. avium Opportunistic, environmental, slow-growing species. Cause
complex subsp. hominissuis cutaneous and disseminated granulomatous disease in
dogs (subsp. hominissuis) and cats (subsp. avium). Some
may cause leprosy syndromes in cats.
Lepromatous mycobacteria M. lepraemurium, the CLG organism, Highly fastidious or unculturable mycobacteria. Cause
and other unnamed species, some nodular granulomatous to pyogranulomatous cutaneous
with genetic resemblance to known disease in cats (all species) or dogs (CLG organism).
nontuberculous mycobacteria
Rapidly growing M. fortuitum, M. smegmatis, Opportunistic, environmental, rapidly growing species.
­nontuberculous M. abscessus, M. chelonae, Cause pyogranulomatous panniculitis, pneumonia, or dis-
­mycobacteria M. ­thermoresistibile, M. goodii, seminated infections in cats or dogs
M. flavescens, M. alvei
Slowly growing M. kansasii, M. ulcerans, Opportunistic, environmental, slowly growing species.
­nontuberculous M. ­genavense, M. malmoense, Cause pyogranulomatous cutaneous or disseminated
­mycobacteria other than M. celatum, M. terrae, M. simiae, disease in dogs or cats.
M. avium ‘M. visibile’*

CLG, Canine leproid granuloma.

*The exact position of ‘M. visibile’ is not clear; it has also been grouped within the lepromatous mycobacteria.

TABLE 44-2
Organisms of the Mycobacterium tuberculosis Complex of Clinical Significance in Dogs and Cats
Major Geographic Distribution
Reservoir of Reported Cases in Human Health
Species Host(s) Hosts Companion Animals Clinical Signs Significance
M. tuberculosis Dogs Humans United States, Africa, Pneumonia and tracheo­ Primary cause of tuber-
southern Europe bronchial lymphadenopa- culosis in humans.
thy; rarely dissemination Reverse zoonosis.
to the CNS, liver, kidney Transmission back to
humans not reported
but may be possible.
M. bovis Cats, rarely Cattle, Southwestern England and Cutaneous lesions, periph- Rare cause of tubercu-
dogs Eurasian Wales, New Zealand, eral lymphadenopathy, losis in humans but
badgers, Argentina, rarely occasionally dissemina- transmission from
other wildlife United States tion to abdominal lymph animals to humans
species nodes and other organs, theoretically possible
osteomyelitis
M. microti Cats, very Voles, wood Southwestern Scotland, Cutaneous lesions, periph- Very rare cause of tu-
rarely dogs mice, shrews, northern and southern eral lymphadenopathy, less berculosis in humans
camelids England, western Europe often pneumonia, arthritis,
osteomyelitis, ocular lesions

whereas the median age of cats infected with M. microti is
8 years.10 All affected cats have been more than 1 year of age,
which likely reflects the long incubation period associated with
these infections. Retroviral infection does not appear to predis-
pose to infection.
M. bovis infections mainly occur in cats that reside in
Wales and southwestern England and that have access to the
outdoors (Figure 44-2).10 Feline M. bovis infections also occur
in New Zealand, where brushtail possums act as reservoir
hosts11; Argentina, where stray cats are often fed raw cattle
420 SECTION 2  Bacterial Diseases
FIGURE 44-1  Geographic distribution of human cases of Mycobacterium tuberculosis infection in the United States, 2005-2011. Compiled from Morbidity and Mortality Weekly reports
data on trends in human tuberculosis, years 2005, 2007, 2008, 2010, and 2011. *The intensity of the red color reflects the absolute number of cases/year, and whether case numbers were
persistently high or varied among the 5 years evaluated. Thus the states that persistently have the highest rates of M. tuberculosis infection are Alaska, Hawaii, California, and New York
(Long Island).
FIGURE 44-2  Geographic distribution of Mycobacterium bovis and Mycobacte-
rium microti isolates from cats in the United Kingdom. (Redrawn from Gunn-Moore DA,
McFarland SE, Brewer JI, et  al. Mycobacterial disease in cats in Great Britain: I. Culture
results, geographical distribution and clinical presentation of 339 cases. J Feline Med Surg
2011;13:934-944.)
lung12; and rarely the United States.13 In the United Kingdom,
spillover of infection from cattle, badgers, and possibly small
rodents to cats is suspected. Aggressive interactions between
cats and infected badgers may also be a route of transmission,
especially as most cutaneous lesions occur at sites where fight
and bite wounds often occur.10,14 Ingestion of unpasteurized
milk by cats is no longer a common route of transmission.
A study from the United States showed no evidence of trans-
mission of M. bovis to cats or dogs that lived on infected
cattle farms.15 Major wildlife reservoirs in North America,
which include white-tailed deer, bison, and elk, are less likely
than small mammals to interact with cats, which may explain
why fewer M. bovis infections are reported in cats from North
America when compared with the United Kingdom. Dogs are
uncommonly infected with M. bovis.16-18 Wounds inflicted
by badgers or a squirrel have preceded disease in affected
dogs.16 In recent years, disease in dogs has been restricted to
southwestern England or Ireland.
M. microti infections mainly occur in cats with outdoor
access from the United Kingdom, with major clusters in south-
western Scotland/northern England and southern England (see
Figure 44-2).10,19 Spillover of disease from voles to cats is sus-
pected.19 Vole and llama genotypes of M. microti have been
identified. Cats in the United Kingdom and one in Switzerland
have been infected with the vole type.20 A dog from France was
infected with the llama type.21
Clinical Features
Signs and Their Pathogenesis
MTBC organisms are inhaled (M. tuberculosis and possibly
M. bovis), ingested (M. bovis or M. microti), or inoculated
into the skin (M. bovis or M. microti) and replicate locally.
Organisms are then ingested by macrophages but survive and
replicate within these cells. Macrophage destruction leads to
recruitment of lymphocytes and additional monocytes, which
initiates tubercle formation. Infected macrophages also travel
to local lymph nodes, with development of a primary complex
lesion. If cell-mediated immunity (CMI) is defective, dissemi-
nation to distant sites can occur. Delayed type hypersensitiv-
ity (DTH) responses can control the initial infection or may
lead to central necrosis and calcification of the primary com-
plex lesions, with persistence of organisms in the center of the
lesions.
Clinical signs in dogs with pulmonary tuberculosis due to
M. tuberculosis are slowly progressive. They consist of chronic,
productive, or nonproductive cough, as well as inappetence,
weight loss, and lethargy.4,7 Rarely, dissemination to nonpul-
monary sites such as the central nervous system (CNS), liver,
 CHAPTER 44  Mycobacterial Infections 421

A B
FIGURE 44-3  Ulcerative skin lesion due to Mycobacterium microti infection in a 6-year-old male neutered domestic shorthair. A, Before treatment. B, After 2 months of treatment with
a combination of marbofloxacin, azithromycin, and rifampin.

or kidney leads to neurologic signs, weight loss, vomiting, or

diarrhea.2,3,9 Involvement of the lungs, liver, kidneys, and/or
lymph nodes can occur in dogs infected with M. bovis.16,18,22
Isolated lung and tracheobronchial node involvement similar to
that often reported for M. tuberculosis was described in one dog
that was infected with M. bovis.18
Most (>75%) cats infected with M. bovis and M. microti
have had cutaneous nodular lesions with or without mandibu-
lar lymphadenopathy. Multifocal peripheral lymphadenopathy
may be present.23 Mesenteric lymphadenopathy, pneumonia,
ocular lesions, and arthritis occur in some cats infected with
M. microti.20,23-25 Dissemination may result in weight loss,
fever, and inappetence. Lung involvement may be associated
with tachypnea, dyspnea, or cough.18

Physical Examination Findings

Dogs with pulmonary involvement caused by M. tuberculo-
sis may show fever, increased lung sounds, and cough. Other
physical examination findings that relate to dissemination of
infection are present less often, such as thin body condition,
neurologic signs, or hepatomegaly. Abnormal findings on
examination of the respiratory tract and/or abdominal organs
may also be detected in dogs with disseminated M. bovis or
M. microti infection. FIGURE 44-4  Mandibular lymphadenopathy in a cat infected with Mycobacterium
The most common physical examination findings in cats microti.
(and to a lesser extent, dogs) that are infected with M. bovis or
M. microti are single or multiple firm cutaneous nodules, and
enlargement of one or more peripheral lymph nodes (Figures Diagnosis
44-3 and 44-4). Cutaneous nodules may be mobile or fixed to
underlying tissues (muscle and bone) and can ulcerate or drain Diagnosis of MTBC infection relies on suggestive history, clinical
purulent fluid. Skin lesions are most prevalent on the head but signs, and radiographic abnormalities, combined with the results
can also occur on the limbs and the trunk.20,23 Enlarged mes- of histopathology, molecular tests, and culture. When mycobacte-
enteric lymph nodes, spleen, and/or liver may be detected on rial infection is suspected, clinical specimens should be divided into
abdominal palpation. Involvement of internal lymph nodes is portions—one for histopathology (in formalin), one for mycobac-
more common with M. bovis infections (21% of cats) than with terial culture, one for routine cultures for other bacterial species,
M. microti infections (9% of cats).10 Lameness may be present and one for mycobacterial PCR (fresh or frozen). The laboratory
in cats with skeletal involvement. should be informed that mycobacterial infection is a possibility.
422 SECTION 2  Bacterial Diseases

A B
FIGURE 44-5  Lateral thoracic radiograph (A) and 7-mm collimated computed tomography (CT) image immediately cranial to the carina (B) from a 9-year-old female spayed golden
retriever with pulmonary tuberculosis. A, Consolidation of the right cranial lung lobe is present together with interstitial to alveolar infiltrates in the right middle lung lobe. B, On the
CT scan, the right cranial lung lobe is consolidated and markedly volume reduced. Air bronchograms are present centrally that extend dorsally toward a focal region of aerated lung. The
granular mineral opacity adjacent to the right margin of the trachea represents mineralized lymphadenopathy of the right tracheobronchial lymph nodes. (From Sykes JE, Cannon AB, Norris
AJ, et al. Mycobacterium tuberculosis complex infection in a dog. J Vet Intern Med 2007;21:1108-1112.)

Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
Laboratory abnormalities in dogs and cats infected with MTBC
organisms are nonspecific. Mild anemia of inflammatory disease
and neutrophilic leukocytosis, sometimes with a left shift, and
monocytosis are often present. The serum chemistry profile may
show hypoalbuminemia and hyperglobulinemia. Hypercalce-
mia of granulomatous disease can also occur in dogs or cats.10,17
Increased serum liver enzyme activities have been reported in
dogs with hepatic involvement.3

Diagnostic Imaging
Plain Radiography
Findings on plain thoracic radiography in dogs infected with
M. tuberculosis or M. bovis include tracheobronchial or medias-
tinal lymphadenopathy,3,4,7 interstitial to alveolar infiltrates,22
pleural effusion,3 and lobar consolidation (Figure 44-5, A).7,22
Similar abnormalities can occur in cats infected with M. microti
and M. bovis (Figure 44-6). Bronchial, alveolar, nodular inter-
stitial, and unstructured interstitial patterns predominate in
cats, and perihilar lymphadenopathy can be present.20,26 Calci-
fication of pulmonary lesions or lymph nodes may be evident in
dogs or cats. Hepatomegaly, splenomegaly, or lymphadenopa-
thy, with or without ascites, may be present on abdominal radi-
ography, or abdominal radiographs may be unremarkable.26
Radiographic evidence of osteomyelitis (especially osteolysis)
can occur in cats infected with M. microti or M. bovis, often in FIGURE 44-6  Pneumonia in a 6-year-old male neutered oriental shorthair with sus-
association with overlying cutaneous lesions.26 pected Mycobacterium microti infection. Bronchoalveolar lavage revealed pyogranuloma-
tous inflammation, but acid-fast stains were negative. Results of an IFN-γ assay suggested
infection by M. microti.
Computed Tomography
Thoracic CT findings have been reported for a dog infected
with M. tuberculosis.7 Lung lobe consolidation, multifo-
cal stellate opacities, bronchial stenosis, bronchiectasis,
 CHAPTER 44  Mycobacterial Infections 423

TABLE 44-3
Assays Available for Diagnosis of Mycobacterial Infections in Dogs and Cats
Assay Specimen Type Target Performance
Acid-fast staining Tissue aspirates, buffy Acid-fast bacteria Insensitive, especially for some mycobacterial
coat smears, body fluids, ­species such as MTBC bacteria. Other bacteria,
airway lavage ­specimens, such as Rhodococcus and Nocardia, may also be
tissues obtained at acid fast, so positive results are not specific for
­biopsy or necropsy ­Mycobacterium spp.
Gamma-interferon Whole blood (­peripheral Cell-mediated Not yet well validated, but show promise for
response assay blood mononuclear ­immune response discrimination between M. bovis and M. ­microti
cells) to ­mycobacterial infections in the United Kingdom. Variable
antigens ­responses to M. avium.
Mycobacterial See acid-fast staining; Culturable Some mycobacterial species are unculturable. Growth
culture avoid use of aspirates or ­Mycobacterium spp. of MTBC organisms and slowly growing nontuber-
swabs if possible culous mycobacteria may take weeks to months,
and identification requires special expertise. Allows
detailed molecular typing and susceptibility testing.
PCR See acid-fast staining Mycobacterial DNA Permits rapid identification of mycobacterial infection
and differentiates MTBC from other mycobacteria,
so appropriate precautions can be instituted. False-
negative results can occur with insufficient specimen
size, if there are very few bacteria present, or if
degradation of DNA occurs. False-positive results
are possible as a result of laboratory contamination.

tracheobronchial wall thickening, and partially mineralized Microbiologic Tests

masses consistent with enlarged mediastinal and tracheobron-
chial lymph nodes were present (Figure 44-5, B). In this dog,
the mineralization was not visible on plain thoracic radio-
graphs. CT in human patients with tuberculosis is considered
more sensitive than plain radiography for detection of pulmo-
nary lesions and enlarged lymph nodes and provides better
visualization of calcifications.
Sonographic Findings
Abdominal sonography in animals with MTBC infections
may be unremarkable or reveal hepatomegaly, splenomegaly,
abdominal lymphadenopathy, and/or ascites. A dog with dis-
seminated M. tuberculosis infection had multifocal hypoechoic
masses within the kidneys and liver and mineralization of the
hepatic and renal parenchyma.4
Cytologic Examination
Cytologic examination of aspirates, effusion, or lavage
specimens from the respiratory tract of dogs with pulmo-
nary tuberculosis typically reveals a mixed population of
often heavily vacuolated histiocytes and smaller numbers
of degenerate or nondegenerate neutrophils and small lym-
phocytes.3,7,22 Intracytoplasmic, nonstaining bacilli were
seen in macrophages in a bronchoalveolar lavage specimen
from a dog infected with M. bovis.22 Moderate anisocytosis
or anisokaryosis of exfoliated respiratory epithelial cells can
be present, which might cause suspicion for pulmonary neo-
plasia.7 The presence of cholesterol crystals, caseous debris,
and concentrically ­laminated crystalline structures known as
calcospherite bodies can be a clue for the presence of MTBC
infection.7,22
Assays available for diagnosis of mycobacterial infections are
listed in Table 44-3.
Acid-Fast Stains
Acid-fast stains can be applied to smears or tissue sections for
detection of tuberculous mycobacteria. Organisms are faintly
beaded bacilli, and are often within macrophages. The presence
of AFB suggests mycobacterial infection, but is not specific for
infection with MTBC organisms. Acid-fast staining of smears or
Cytospin preparations can be insensitive for detection of MTBC
organisms, and negative results do not rule out infection.
Immunologic Assays
Intradermal inoculation of tuberculin (a purified protein deriva-
tive from M. tuberculosis), or the Mantoux test, is well vali-
dated and widely used in human medicine for diagnosis of, and
screening for, infection with MTBC organisms. Infected individ-
uals develop a cutaneous DTH reaction at the site of inoculation
48 to 72 hours later. False-negative test results can occur as a
result of underlying immunosuppression. False-positive results
occur as a result of nontuberculous mycobacterial infection.
Cats do not respond reliably to tuberculin skin testing. In dogs,
tuberculin testing has been performed on the medial surface of
the pinna, but false-positive results have been documented with
a variety of other bacterial infections. Tests for anti-mycobacte-
rial antibodies are also not specific.27 Cell-based assays (ELISA
and ELISPOT), which measure IFN-γ production by patient
peripheral blood mononuclear cells in response to specific myco-
bacterial antigens, have been used for diagnosis of tuberculosis
in humans and cattle. These assays have been adapted for use in
cats and have the potential to rapidly distinguish infection with
424 SECTION 2  Bacterial Diseases
M. bovis from that with M. microti when responses to different
mycobacterial antigens are compared.28
Bacterial Isolation and Identification
Routine aerobic, anaerobic, fungal, and mycoplasma cultures
are negative when performed on specimens collected from ani-
mals with MTBC infections. Mycobacterial culture is required
and is the gold standard for diagnosis of MTBC infection. The
use of laboratories with expertise in isolation of MTBC is rec-
ommended. Respiratory specimens such as respiratory lavages
are usually treated with the mucolytic N-acetylcysteine and a
sodium hydroxide solution to destroy contaminating bacteria.
Specimens from normally sterile sites, such as pleural fluid, are
not decontaminated so that viable mycobacteria are preserved
as much as possible. Specimens are then inoculated onto spe-
cial liquid or solid media for isolation of mycobacteria, such as
7H11 agar or Lowenstein-Jensen media (an egg-based medium).
Modifications to the media composition may be required on the
basis of the mycobacterial species suspected, so good commu-
nication between the clinician and the microbiology laboratory
can optimize the chance of successful culture. Growth is evident
1 to 3 weeks after inoculation of liquid media, and 3 to 8 weeks
after inoculation of solid media. However, inoculation of solid
media is still required, because some strains grow only on solid
media, and growth on solid media allows assessment of colony
morphology. Once growth is evident, nucleic acid–based assays,
mycolic acid analysis with high-performance liquid chromatog-
raphy, or mass spectrometry (MALDI-TOF)29 can be used to
determine whether the organism belongs to the MTBC. Antimi-
crobial drug susceptibility testing can also be performed.
Currently, performance of a series of phenotypic (biochemi-
cal) assays on cultured isolates is the gold standard method
for differentiation of mycobacterial species that belong to the
MTBC. Molecular techniques such as PCR-restriction endonu-
clease analysis, DNA probing, and 16S rRNA gene sequencing
do not differentiate among MTBC species. Specialized molec-
ular techniques for further typing of MTBC isolates include
mycobacterial interspersed repetitive unit-variable-number
tandem repeat (MIRU-VNTR) typing and spoligotyping. Spo-
ligotyping is a PCR-hybridization typing method that targets
the direct repeat region of MTBC strains. This region contains
multiple DNA sequence repeats interspersed with nonrepetitive
spacer sequences. MTBC species vary in the numbers of repeats
and the presence or absence of some spacers and can be dif-
ferentiated on the basis of their hybridization patterns. Spoli-
gotyping also allows identification of prevalent M. tuberculosis
genotypes, such as the Beijing genotype, an aggressive, highly
transmissible strain that has been associated with multiple drug
resistance. MIRU-VNTR is often combined with spoligotyping
for identification of MTBC organisms. Spoligotyping has been
performed on several MTBC isolates from dogs and cats.
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR-based assays are available on a commercial basis
in some countries for rapid detection of Mycobacterium spp.
DNA in clinical specimens from dogs and cats. An understand-
ing of the range of mycobacterial species detected by an assay
is of paramount importance for proper interpretation of the
results. Genus-specific Mycobacterium spp. assays are available,
as well as assays that detect mycobacteria other than tuberculo-
sis (MOTT) organisms. These assays can be used in combination
to determine whether an MTBC organism is present. A positive
result with a genus-specific assay but a negative result with an
assay for MOTT organisms implies the presence of an MTBC
organism. The results of mycobacterial PCR assays should be
interpreted in light of clinical findings, and PCR assays should
always be used with mycobacterial culture, which is the only
way to confirm the mycobacterial species involved and allows
antimicrobial drug susceptibility testing. The latter is important
given the public health implications of MTBC infections.
Pathologic Findings
Gross Pathologic Findings
The most frequent gross pathologic findings in dogs infected
with M. tuberculosis are pulmonary congestion, lobar consoli-
dation, or granulomatous mass formation and sometimes mas-
sive enlargement of tracheobronchial and mediastinal nodes.
Lymph nodes may contain multifocal gritty, yellow, caseous
material.4,7,22 Pleural effusion may also be present.3 In dogs
with disseminated disease, pale nodular lesions may be found
in abdominal organs such the liver, peritoneal surfaces, and kid-
neys.2,4,22 In one unusual case, a dog from Africa had nodular
lesions that were confined to the liver and brain.2 Dogs infected
with M. bovis have abnormalities similar to those infected
with M. tuberculosis.16-18 In addition to cutaneous lesions and
peripheral lymphadenomegaly, cats with disseminated M. bovis
or M. microti infections may have abdominal lymphadeno-
megaly and multifocal tumor-like, grayish-white masses within
parenchymal organs that can have hemorrhagic margins and/or
a soft purulent center. Pulmonary lesions are often grayish red
and may be accompanied by serosanguineous pleural effusion.
Histopathologic Findings
Histopathologic findings are similar for all MTBC diseases.
Characteristic tubercles or granulomas are present in tissues,
which vary in size and may coalesce (Figure 44-7). Tubercles
consist of abundant epithelioid macrophages and lesser num-
bers of neutrophils. Multinucleated giant cells are rarely present
in dogs or cats.4,30 The inflammatory foci are surrounded by
a layer of macrophages, neutrophils, lymphocytes, and plasma
cells and an outer layer (or background) of granulation tissue.
The center of the tubercle may be necrotic. Central mineraliza-
tion and lipid accumulation occurs in some dogs infected with
M. tuberculosis,7,22 but mineralization is rare in cats infected
with M. microti or M. bovis. Acid-fast stains reveal low num-
bers of AFB in tubercle centers in most cases, but a careful
search may be required (Figure 44-8).4,7
Treatment and Prognosis
Dogs and cats with suspected MTBC infection should be placed
in isolation, and aerosol precautions are indicated (see Chapter
11). Treatment of dogs and cats with MTBC infection is contro-
versial because of concerns that relate to zoonotic potential. It
should only be performed after owners have been fully educated
about the potential risks of disease transmission. Most affected
dogs have been euthanized without specific treatment, but occa-
sionally euthanasia of animals with non-MTBC mycobacterial
infection has occurred before a diagnosis of MTBC was con-
firmed. Combination drug therapy with the antimycobacterial
drugs ethambutol, isoniazid, rifampin, and pyrazinamide is used
to treat human patients. Ethambutol and isoniazid interfere with
mycobacterial cell wall synthesis; the mechanism of action of
pyrazinamide is not known, and M. bovis is naturally resistant

A B
FIGURE 44-7  Multifocal granuloma formation in the lung (A) and thoracic lymph node (B) of a 9-year-old female spayed golden retriever with pulmonary tuberculosis. The granu-
loma within the lymph node contains a lake of lipid and necrotic cellular debris, surrounded by an internal layer of epithelioid macrophages, a thick layer of fibrosis, and an external layer of
lymphocytes, plasma cells, and histiocytes. Fite and Ziehl-Neelsen acid-fast stains revealed scant numbers of acid-fast positive bacteria in areas of central necrosis (not shown).
FIGURE 44-8  Rare acid-fast bacteria in a biopsy specimen from a cat infected with
M. microti.
to it. Treatment of a dog with M. tuberculosis infection was
attempted with rifampin, isoniazid, and clarithromycin, but the
dog was euthanized after seizures developed.7 The seizures were
suspected to be an adverse effect of isoniazid administration.
Successful treatment of cats with cutaneous and/or pulmo-
nary MTBC infections has been achieved with combinations of
fluoroquinolones, clarithromycin or azithromycin, doxycycline,
and a rifamycin; most successfully treated cases have received
a fluoroquinolone, clarithromycin or azithromycin, and a rifa-
mycin (Table 44-4). Surgical excision of small skin lesions can
be curative if dissemination is not present, but lesions may
dehisce and progress if resection is not complete, and it may
not always be possible to determine whether dissemination has
occurred. The use of three drugs has been recommended for the
first 2 months of treatment (e.g., rifampin, clarithromycin, and
CHAPTER 44  Mycobacterial Infections 425
a fluoroquinolone), followed by a continuation period with two
drugs (usually the macrolide and the fluoroquinolone, because
rifamycins are more often associated with adverse effects).
Esophagostomy tube placement may be necessary to facilitate
treatment and reduce the chance of exposure of the owner to the
infection through bite or scratch wounds.
Immunity and Vaccination
There are no vaccines available for prevention of mycobacterial
disease in animals.
Prevention
Prevention of M. tuberculosis infections in dogs relies on appro-
priate control and treatment of M. tuberculosis infections in
affected humans that are in close contact with dogs. Prophylaxis
with a single antimycobacterial drug such as isoniazid could be
considered for dogs that reside with affected humans, but iso-
niazid has the potential to cause adverse effects such as seizures
in dogs. The possibility of chronic infection by potentially zoo-
notic pathogens such as M. tuberculosis should be considered
before stray dogs are adopted from underdeveloped countries.
In cats, prevention of M. bovis and M. microti infections can
be achieved when cats are housed indoors and rodent popula-
tions controlled. The feeding of raw cattle or game (e.g., deer,
elk) also has the potential to transmit M. bovis to dogs or cats
and should be discouraged.
Public Health Aspects
Disease caused by MTBC bacteria is usually notifiable to pub-
lic health authorities. More than 90% of human tuberculosis
is caused by M. tuberculosis. Only 1% of cases result from
M. bovis infection, and human infection with M. microti is very
rare.31 Factors such as HIV infection, homelessness, and intra-
venous drug use predispose humans to M. tuberculosis infec-
tion. Tuberculosis continues to be an important public health
problem worldwide and especially in Africa. It is second only
to HIV as a cause of death worldwide from a single infectious
426 SECTION 2  Bacterial Diseases

TABLE 44-4
Suggested Drug Doses for Treatment of Mycobacterial Infections*
Interval
Drug Dose (mg/kg) Route (hours) Major Adverse Effects in Dogs and Cats
Isoniazid 5-10 PO 24 Hepatotoxicity, seizures. Start at a low dose; do not exceed
300 mg/day. Monitor liver enzymes.
Ethambutol 10-25 PO 48-72 Unknown; neuropathies, especially optic neuritis with visual
disturbances occur in humans.
Rifampin 5-10 PO 12-24 Gastrointestinal, hepatotoxicity, orange discoloration of urine,
tears, sclera, mucous membranes, feces. Monitor liver enzymes.
Clarithromycin 7.5 PO 12 Rare; hepatotoxicity and gastrointestinal signs possible.
Doxycycline 10 PO 12 Gastrointestinal
Enrofloxacin 5-10 (dogs only)† PO 24 Gastrointestinal
Marbofloxacin 2.75-5.5 PO 24 Gastrointestinal
Clofazimine 25 mg/cat, 4-8 mg/kg PO 24 Cutaneous photosensitization,‡ orange pigmentation to the skin,
(dogs) gastrointestinal. May be difficult to obtain in some countries.

*See text for recommended drug combinations for each mycobacterial species and additional drugs that may be useful.
†The use of other fluoroquinolones such as marbofloxacin or pradofloxacin is preferred in cats because of the risk of retinal toxicity (see Chapter 8).
‡Bennett S. Photosensitisation induced by clofazimine in a cat. Aust Vet J 2007;85:375-380.

agent,32 and drug resistance is emerging globally. Over the past
2 decades, case numbers have decreased to the lowest levels
in history in the United States.33 In the United States, the inci-
dence rate is highest among non-Hispanic Asians, non-Hispanic
blacks, and Hispanics, which is 25, 8, and 7 times greater,
respectively, than the rate for non-Hispanic whites.33 Disease
in immigrants to the United States results from reactivation of
disease acquired in other countries.
Public health authorities should be notified if a diagnosis
of MTBC infection is made in a dog or cat. An investigation
may be performed by these authorities in order to identify the
source of infection, and it may be necessary to perform one
or more Mantoux tests on individuals with significant contact
with infected animals. Although transmission of M. tuberculo-
sis from infected dogs to humans has not been reported as a
result of direct contact, veterinary staff were infected when a
necropsy of an infected dog was performed, possibly following
aerosolization of infected brain tissue when the skull was sec-
tioned with an electric saw.2 Properly fitted high-density surgi-
cal masks (N95 masks) should be worn if surgery or necropsy is
performed on a dog or cat suspected to have tuberculosis.
Mycobacterium avium Complex
Etiology and Epidemiology
The Mycobacterium avium complex (MAC) consists of two
closely related species, M. avium and Mycobacterium intra-
cellulare. There are four subspecies of M. avium: hominissuis,
avium, paratuberculosis, and silvaticum. M. avium subsp.
hominissuis causes most human infections. The distribution
of subspecies in affected dogs and cats is currently not clear,
because molecular methods are required for identification
at the subspecies level. M. avium subsp. hominissuis infec-
tions have been identified in several dogs from continental
Europe,34,35 whereas M. avium subsp. avium was isolated from
an affected cat.36 The DNA of M. avium subsp. paratuberculo-
sis was detected in intestinal biopsies from dogs with chronic
gastrointestinal signs, but the role of this organism in disease
was not clear.37 Unlike MTBC organisms, which are associ-
ated with reservoir hosts, MAC organisms are environmental
saprophytes with a worldwide distribution. They can be found
in soil, dust, and aquatic environments that include showers,
faucets, household drinking water, swimming pools, and hot
tub spas.38 The organisms also infect free-living amoebae and
form biofilms, which may promote environmental persistence.
In the United Kingdom, a spatial cluster of infection has been
identified in cats from eastern England.10 Subclinical infections
with M. avium subsp. avium are common among birds, and
M. avium subsp. avium is the main cause of avian ­tuberculosis.39
Ingestion of infected birds or bird feces may be a route of trans-
mission to cats or dogs, but this requires clarification.
MAC organisms cause opportunistic infections in immu-
nocompromised individuals. Most affected dogs and cats are
1 to 5 years of age. In dogs, disease occurs sporadically in any
breed, but basset hounds, miniature schnauzers, and possibly
Yorkshire terriers may be predisposed, possibly because of an
inherited CMI deficiency. In cats, retrovirus infection or treat-
ment with potent immunosuppressive drugs may predispose to
MAC infection, although most cats have no identifiable immu-
nodeficiency. Siamese cats as well as Abyssinians and Somalis
may be predisposed.40,41 Disseminated MAC infection has also
been reported in feline renal transplant recipients42,43 and in
a cat treated with chemotherapy for lymphoma.44 Infection is
not transmissible from one infected individual to another, but
disease may initially be indistinguishable from that caused by
MTBC bacteria. In the United States, although still rare, infec-
tion of cats and dogs with MAC organisms is more common
than infection with MTBC organisms. In the United Kingdom,
M. avium accounted for only 15% of 159 culturable mycobac-
terial infections in cats (73% were MTBC infections).10 The
median age of affected cats is 8 years.

FIGURE 44-9  Five-year-old female spayed husky dog with Mycobacterium avium
lymphadenitis. Swellings in the mandibular region are apparent.
Clinical Features
Signs and Their Pathogenesis
Disease due to MAC organisms occurs when organisms in
soil, water, or dust are inhaled, ingested, or inoculated into
skin wounds in the face of host immunocompromise. Dis-
seminated infection often leads to nonspecific signs such as
fever, lethargy, inappetence, and weight loss, together with
signs that relate to specific organ involvement. In cats, dis-
ease resembles that caused by M. bovis or M. microti, with
cutaneous lesions (especially on the head and limbs) in some
cats, osteomyelitis, pulmonary involvement with tachypnea
or cough, peripheral and abdominal lymphadenomegaly, and
gastrointestinal, liver, splenic, renal, omental, and uncom-
monly CNS or marrow involvement.41,42,44-50 Gastrointesti-
nal involvement may initially manifest as weight loss despite
a good appetite.41
Dogs with MAC infections often develop marked peripheral
or abdominal lymphadenopathy, tonsillar enlargement, hepato-
splenomegaly, and/or osteomyelitis. In some dogs, mandibular
or cervical lymphadenopathy may be so severe that respiratory
distress and dysphagia occur (Figure 44-9).51 At necropsy, AFB
and associated inflammatory lesions may also be found in the
small and large intestines, liver, spleen, lung and pleura, bone
marrow, and rarely the spinal cord.34,52-56 Involvement of the
gastrointestinal tract may lead to chronic vomiting, diarrhea,
hematochezia, melena, inappetence, and weight loss. Intesti-
nal rupture in one dog led to bacterial peritonitis. Even though
inflammatory lesions with intralesional AFB have been detected
in the lung at necropsy, pulmonary involvement is not a promi-
nent clinical feature of MAC infections in dogs, which may help
to distinguish the disease from M. tuberculosis infection.
Physical Examination Findings
Physical examination of cats with MAC infections may reveal
fever, a thin body condition, cutaneous lesions similar to
those described for MTBC infection, peripheral or abdominal
CHAPTER 44  Mycobacterial Infections 427
FIGURE 44-10  Digital osteomyelitis in an 8-year-old female spayed Yorkshire terrier
dog with Mycobacterium avium infection. The first and second phalanges of the fifth digit
are destroyed, and there is irregular mineralization within a large soft tissue swelling in
this region. There is also moderate soft tissue swelling at the second digit. The third and
fourth digits had been previously amputated at the mid-metacarpal bones, which are now
fused at their distal aspect.
lymphadenopathy, renomegaly, hepatomegaly, tachypnea, and/
or increased lung sounds. Rarely, neurologic signs such as nys-
tagmus occur.41 In dogs, low-grade fever, a thin body condition,
dehydration, mucosal pallor, tonsillar enlargement, lymphad-
enomegaly (especially of the mandibular or abdominal nodes),
hepatosplenomegaly, and/or abdominal pain may be found.
Melena or hematochezia may be detected on rectal examina-
tion. Osteomyelitis may be associated with lameness or mass
lesions in association with underlying bone involvement (Fig-
ure 44-10). A dog with spinal cord involvement had pelvic limb
paresis.53
Diagnosis
Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
Findings on routine laboratory tests in dogs and cats with MAC
infections are similar to those described for MTBC infection.
Most affected dogs or cats have had mild to moderate nonre-
generative anemia, neutrophilia with bandemia, lymphopenia,
hypoalbuminemia, hyperglobulinemia or hypoglobulinemia,
and sometimes hypercalcemia, hyperbilirubinemia, and
increased liver enzyme activities.48,54,57 An absence of hemato-
logic or biochemical abnormalities has also been described.
Diagnostic Imaging
Plain Radiography
When radiographic abnormalities are present in cats infected
with M. avium, they often consist of diffuse interstitial or
bronchointerstitial pulmonary patterns.41,48 Diffuse interstitial
patterns may be present in the absence of clinical signs of respi-
ratory involvement. Thoracic lymphadenopathy or mediastinal
428 SECTION 2  Bacterial Diseases
FIGURE 44-11  Aspirate cytology from a cutaneous mass in a cat that developed
after prolonged (>1 year) treatment with cyclosporine. Large numbers of negatively
stained mycobacterial organisms are present. (Image courtesy Dr. William Vernau, Univer-
sity of California, Davis Veterinary Clinical Pathology Service.)
masses may be present in dogs.35 Abdominal radiographs may
show abdominal masses or hepatosplenomegaly in both dogs
and cats. Radiographs of affected bone may reveal soft tissue
swelling and bony lytic lesions or periosteal proliferation (see
Figure 44-10).
Sonographic Findings
Abdominal sonography in animals with MAC infections
may be unremarkable or reveal hepatomegaly, splenomegaly,
hypoechoic nodules within the liver or spleen, intestinal wall
thickening, hyperechoic mesentery, or enlarged, hypoechoic, or
heteroechoic abdominal nodes.58
Cytologic Examination
Cytologic examination of aspirates from lymph nodes, abdomi-
nal organs, or respiratory lavage specimens from cats or dogs
with M. avium infection reveals large numbers of histiocytes
with nonstaining intracellular bacteria (Figure 44-11) and lower
numbers of neutrophils.
Microbiologic Tests
For assays available for diagnosis of mycobacterial infections in
dogs and cats, see Table 44-3.
Acid-Fast Stains
Application of acid-fast stains to smears or tissue sections can be
used to identify MAC organisms in clinical specimens. In gen-
eral, AFB are identified more readily in MAC infections when
compared with MTBC infections, and most reports of affected
dogs and cats report the detection of organisms in cytology
specimens stained with acid-fast stains.
Bacterial Isolation and Identification
See the previous discussion of MTBC infection for informa-
tion on mycobacterial culture. Like MTBC organisms, MAC
organisms grow slowly in culture. Up to 6 weeks of incuba-
tion may be required for visible growth. MAC species are then
identified using commercial DNA probe-based assays or PCR
analysis. Subspecies determination requires specialized typing
techniques such as IS1245 restriction fragment length poly-
morphism typing, MIRU-VNTR, or the use of PCR assays that
detect gene fragments present in some M. avium subspecies but
not others.34,36,59
Molecular Diagnosis Using the Polymerase Chain Reaction
See the previous discussion of MTBC infection for information
on mycobacterial PCR assays.
Pathologic Findings
In general, pathologic findings in cats or dogs with MAC infec-
tion consist of focal, multifocal, or generalized lymphadeno-
megaly (up to 5 cm in diameter), and nodules in the spleen,
liver, lungs, omentum, intestinal wall, and occasionally the kid-
neys. Nodules are white, gray, or yellow and may be caseous
on cut section. Gastrointestinal mucosal hemorrhage may be
evident in dogs.54
Histopathology in MAC infections typically reveals large
numbers of histiocytes and lesser numbers of neutrophils, which
in some cases surround necrotic foci. Multinucleated giant cells
are rarely seen, and mineralization has not been described.
Moderate to large numbers of AFB are usually present within
histiocytes (Figure 44-12).
Treatment and Prognosis
Treatment of MAC infections in dogs or cats is challenging,
but apparent cure has been reported in a few cats.36,41,48 As
for infection with MTBC organisms, the use of two or three
drugs in combination is recommended to prevent emergence of
resistance. The availability of macrolides (especially clarithro-
mycin) has greatly improved outcome for human patients with
MAC infections, so inclusion of clarithromycin or azithromycin
is important. The currently recommended treatment for humans
with MAC infections consists of a ­rifamycin, ethambutol, and
a macrolide such as clarithromycin. Treatment of a dog with
a combination of drugs that did not include a macrolide was
unrewarding.51 Various ­combinations of clarithromycin, clo-
fazimine, doxycycline, rifamycins, and fluoroquinolones have
been used to treat affected cats (see Table 44-4). Clofazimine
is an ­antimycobacterial drug with an unknown mechanism of
action. In human patients, it has primarily been used to treat
lepromatous or rapidly growing opportunistic mycobacteria,
but may have some activity against MAC organisms. It may be
difficult or impossible to obtain in some countries.
Public Health Aspects
Like dogs and cats, humans acquire MAC infections from the
environment; direct transmission from animals to people has
not been described. Ownership of caged birds by immunocom-
promised humans has been discouraged in the past because of
the potential that these birds might shed MAC organisms. How-
ever, human disease is caused by M. avium subsp. h ­ ominissuis
and not the avian organism (subsp. avium), so the feces of
caged birds may not represent a significant source of infection.
Three clinical forms of MAC disease occur in people: local-
ized pulmonary disease (which includes “hot tub lung disease,”
a hypersensitivity pneumonitis), cervical lymphadenitis, and
disseminated disease.60 Disseminated disease occurs most
often in HIV-infected humans, but children with congenital

A B
FIGURE 44-12  A, Granulomatous inflammation in the brain of a 10-year-old male neutered Siamese mix cat with disseminated Mycobacterium avium infection. H&E stain. B, Section
from the same cat stained with Ziehl-Neelsen (acid-fast) stain. There are large numbers of intracellular acid-fast bacteria.
immunodeficiencies can also be affected. Prophylactic antimyco-
bacterial drug treatment is recommended for HIV-infected humans
with CD4+ T cell counts that are less than 50 cells/mm3.60 Cervi-
cal lymphadenitis is an ­uncommon disease of children less than
3 years of age.
Although transmission of MAC organisms from diseased
dogs and cats to humans should not, in theory, occur by direct
contact, it may not be possible to distinguish infection with
M. avium from infection with MTBC organisms (especially
M. bovis) based on clinical presentation. Thus, in regions where
M. bovis infections occur, animals suspected to have MAC
infections should be isolated until the results of PCR assays con-
firm MAC infection. Cytologic or histopathologic evidence of
large numbers of organisms also suggests MAC as opposed to
MTBC infection. Veterinary staff should take care to avoid nee-
dle-stick injuries or cutaneous inoculation when collecting spec-
imens (such as lymph node aspirates) from dogs and cats with
M. avium infections because of the possibility that disease might
occur after cutaneous inoculation. Sedation is recommended
whenever lymph node aspiration is indicated for an animal with
an unknown cause of lymphadenopathy. When mycobacterial
disease is suspected, care should also be taken not to aerosolize
tissues at necropsy (as occurs with electric saws).
Lepromatous Mycobacteria
Etiology and Epidemiology
Lepromatous mycobacteria are highly fastidious or uncultur-
able mycobacteria that cause nodular cutaneous lesions in dogs
or cats. Feline leprosy is caused by Mycobacterium lepraem-
urium and probably several other mycobacterial species. In fact,
with the recent discovery of these other mycobacterial species,
localized nodular cutaneous disease in cats caused by MAC
organisms, or organisms that resemble other nontuberculous
mycobacteria (NTM), has fallen under the general heading of
feline leprosy syndromes.61-63
Feline leprosy has been described in the Netherlands, the United
Kingdom, the United States, western Canada, Australia, New
Zealand, Italy, and Greece. Affected cats range in age from 1 year
to 14 years and are often from rural or semirural environments.
CHAPTER 44  Mycobacterial Infections 429
Inoculation of organisms into the skin through rodent bites or cat
fight wounds is the suspected mode of transmission.
Disease in dogs is known as canine leproid granuloma syn-
drome (CLGS). It has mainly been reported from the western
United States and coastal regions of Australia.64-66 The organ-
ism that causes CLGS has only been partially characterized by
the use of PCR sequencing. It has never been isolated in culture
and does not yet have a Latin name, but is often referred to as
the “CLG organism.” A similar, and possibly the same, organ-
ism has also been detected using PCR in cats with leprosy.63
Boxer dogs or their crosses are predisposed, and possibly also
German shepherd dogs.65,66 The mode of transmission to dogs
is unknown. Transmission by biting insects has been hypoth-
esized because most lesions occur on the pinnae and head.
Clinical Features
Lepromatous mycobacteria cause single or multiple nodular
cutaneous or subcutaneous lesions, which may ulcerate. In cats,
lesions are usually on the head or forelimbs, but can also occur at
other body sites. Affected cats are usually systemically well. Two
pathologic variants of feline disease have been described.62,63
The first form, “tuberculoid feline leprosy,” is characterized
by multifocal to coalescing pyogranulomas with central case-
ous necrosis, as well as increased numbers of lymphocytes and
plasma cells in the dermis and panniculus. Multi­nucleate giant
cells may or may not be present, and acid-fast stains reveal
variable numbers of AFB, primarily in regions of necrosis. The
second form, “lepromatous feline leprosy,” is characterized by
pyogranulomatous dermatitis and panniculitis with sheets of
epithelioid macrophages and neutrophils, moderate to abundant
multinucleate giant cells, and large numbers of AFB with mini-
mal necrosis. Organisms that resemble M. lepraemurium can be
detected in both these clinical forms, whereas the CLG organism
has only been detected in cats with the tuberculoid form.63 The
distribution of histopathologic types may vary geographically.
In dogs, CLGS is characterized by the development of firm
cutaneous nodules that range in diameter from millimeters to
several centimeters. Larger lesions may ulcerate. Most lesions
are found on the pinnae, but they may also be found on the head,
muzzle, forelimbs, and trunk (Figure 44-13). Histopathology
430 SECTION 2  Bacterial Diseases

B C
FIGURE 44-13  A, Pinna of a 5-year-old male neutered boxer dog with CLGS. A crusted lesion was present that measured 1 × 2 cm in diameter. B, Histopathology of CLGS lesions
reveals pyogranulomatous inflammation. C, A BCG immunostain (brown) identified the presence of intracellular Mycobacterium spp. organisms.

reveals coalescing accumulations of histiocytes and neutrophils,
with fewer numbers of lymphocytes and plasma cells.64,66 Mul-
tinucleate giant cells may be present, and there may be large
numbers of intracellular AFB.
Diagnosis
lesions, but has the potential to be followed by dehiscence and
extension of disease. If necessary, antimicrobial drug treatment
for several weeks followed by surgery may be most successful.
In dogs, granulomas may resolve spontaneously within 1 to
3 months. In dogs with progressive or widespread CLGS, a com-
bination of clarithromycin and a rifamycin may be effective.67

Diagnosis of leproid syndromes is based on the presence of typi-
cal skin lesions, absence of signs of dissemination or systemic
illness, suggestive cytologic and histopathologic findings, nega-
tive mycobacterial culture results, and positive 16S rRNA gene
PCR assay results where PCR assays are available. Cytologic
examination of fine-needle aspirates usually reveals pyogranulo-
matous inflammation with intracellular, negatively stained bac-
teria, which subsequently stain with acid-fast stains. Sequence
analysis of PCR products that are generated from biopsy speci-
mens is required to determine the likely species present.
Treatment
Treatment of feline leproid lesions with a combination of clo-
fazimine and clarithromycin is often successful (see Table 44-4).
Wide surgical excision could be considered for small, localized
Prevention and Public Health Aspects
Mycobacterial organisms that cause canine and feline leproma-
tous disease are not transmitted directly to humans. However,
lesions may resemble those caused by M. microti and M. bovis,
so where these diseases are endemic, precautions should be taken
until a causative organism is identified with molecular methods.
Nontuberculous Bacteria Other Than
Mycobacterium avium-intracellulare
Etiology and Epidemiology
NTM species include a variety of opportunistic, environmen-
tal mycobacteria that have been classified based on their rate
 CHAPTER 44  Mycobacterial Infections 431

TABLE 44-5
Cutaneous and Disseminated Disease in Dogs and Cats Caused by Slow-Growing Opportunistic Nontuberculous Mycobacteria
Mycobacterial Species ­
Species Affected Clinical Abnormalities Geographic Location
M. kansasii Dog Chronic pleural effusion with pulmonary arterial and pleuro- North Carolina, USA83
peritoneal shunt infection
M. simiae Cat Disseminated disease with generalized lymphadenopathy, lung Switzerland84
and skin involvement, and granulomatous chorioretinitis
M. genavense Cat (FIV+) Disseminated disease with involvement of lungs, liver, lymph New South Wales, Australia85
nodes, skin, kidney, spleen
Dog Disseminated disease with involvement of lymph nodes and New York, USA86
possibly liver and spleen
M. xenopi Cat Cutaneous lesion Australia87
Cat Disseminated disease with kidney, lymph node, pancreatic, Florida, USA88
and marrow involvement
Cat Disseminated disease with lymphadenopathy, gastrointestinal Wisconsin, USA89
involvement, and ascites
M. xenopi-like Cat (FIV+) Tracheal granuloma Italy90
M. ulcerans Dog Cutaneous ulcerating lesions on distal limbs or tail base Victoria, Australia91
Cat Mass on nasal bridge Victoria, Australia92
‘M. visibile’ Cat Cutaneous and disseminated disease with involvement of the Western Canada and United
(­originally eyes, skeletal muscle, and a variety of other organs except States93
‘M. visibilis’) the liver and kidneys
M. malmoense Cat Cutaneous involvement United Kingdom10
M. celatum Cat Cutaneous involvement and fever United Kingdom10
M. terrae Cat Cutaneous involvement United Kingdom94

of growth in culture (rapid, intermediate, or slow) (see Table
44-1). They are found in soil and a variety of water sources and
cause cutaneous, pulmonary, or disseminated disease in dogs
and cats. Rapidly growing mycobacteria (RGM) grow in cul-
ture within 7 days and include several groups of organisms to
which M. fortuitum, M. septicum, M. abscessus, M. chelonae,
M. smegmatis, M. thermoresistibile, M. goodii, and M. muco-
genicum belong. Slowly growing mycobacteria require more
than 7 days (and sometimes many months) before significant
growth occurs in culture. Other than MAC organisms, exam-
ples include M. kansasii, M. terrae, M. xenopi, M. ulcerans,
M. malmoense, M. simiae, and M. genavense.
M. goodii.72-75 No age, breed, or sex predisposition has been
recognized, but most dogs are in good body condition or obese.
Some dogs have a history of bite wounds or trauma.73 Concur-
rent hyperadrenocorticism was described in one dog.75
M. fortuitum can cause pulmonary disease in dogs76-79 as
well as bacteremia.72 Lipoid mycobacterial pneumonia rarely
occurs in cats and may arise in association with lactulose or par-
affin treatment for furballs.80 Pneumonia was also described in
a cat infected with M. thermoresistibile.81 Disseminated RGM
infections are very rare. Disseminated disease that resembled
MAC infection was described in a basset hound infected with
M. smegmatis.82

Rapidly Growing Mycobacteria Slowly Growing Mycobacteria

RGM are responsible for the vast majority of cutaneous myco-
bacterial disease in cats in the United States and Australia, but
infections have also been described in the United Kingdom,
Canada, western Europe, and New Zealand. The most com-
monly isolated species in both the United States and Australia
are M. fortuitum and M. smegmatis, but infections with other
RGM also occur (see Table 44-1).68-71 These organisms have a
tropism for adipose tissue, and when inoculated into the skin
by means of cat fight injuries (bites or scratches) or other pen-
etrating wounds (and rarely surgical wounds),72 mycobacterial
panniculitis develops. Most affected cats are female spayed cats.
Underlying immunosuppressive disease is usually not present.
Cutaneous disease occurs less often in dogs and results from
infection with M. fortuitum, M. smegmatis, M. abscessus, or
A variety of slowly growing mycobacteria cause cutaneous or dis-
seminated disease in dogs and cats that resembles disease caused
by MAC organisms (Table 44-5).83-94 These include the known
human pathogens M. kansasii, M. simiae, M. genavense, M.
xenopi, M. celatum, M. malmoense, M. terrae, and M. ulcerans.
Some, but not all affected cats have been infected with FIV, and
one cat had CD4+ lymphopenia.88 M. ulcerans causes ulcerative
skin lesions in humans, cats, or dogs in certain parts of Australia.
Clinical Features
Rapidly Growing Mycobacteria
In cats, mycobacterial panniculitis occurs most often in the
inguinal fat pad, but lesions may also be found elsewhere
432 SECTION 2  Bacterial Diseases
on the lateral abdomen, perineum, dorsum, and pelvic limbs
(Figure 44-14). The subcutis becomes thickened and adheres to
the dermis, after which nodular and draining skin lesions develop.
The majority of affected cats are otherwise well; the development
of systemic illness is rare. In dogs, nodular lesions or nonhealing
wounds with draining tracts often occur in the inguinal region,
cervical region, and/or other proximal body locations.
Pneumonia due to RGM generally results in signs of fever,
lethargy, decreased appetite, cough, and tachypnea, with
increased lung sounds on thoracic auscultation. Thoracic radio-
graphs frequently reveal lung lobe consolidation and alveolar
patterns. In the cat with M. thermoresistibile pneumonia, exten-
sive pulmonary mineralization developed.81 The dog with dis-
seminated M. smegmatis infection had granulomatous hepatitis
and lymphadenitis.
Slowly Growing Mycobacteria Other Than MAC
Clinical features of slowly growing mycobacterial infections
other than MAC infections have varied from one case to another
and are summarized in Table 44-5. Animals with disseminated
disease can have clinical abnormalities that are indistinguish-
able from those caused by M. avium.
Diagnosis
Diagnosis of RGM infection is usually based on the presence of
consistent clinical abnormalities, and the results of cytology, his-
topathology, and mycobacterial culture (see Table 44-3). Cyto-
logic examination of fine needle aspirates or lavage specimens
generally reveals pyogranulomatous inflammation. However,
airway lavage fluid from a dog with M. fortuitum pneumonia
A C
FIGURE 44-14  Cutaneous-subcutaneous infections with rapidly-growing mycobacteria. A and B, Thirteen-year-old male neutered Siamese cat with mycobacterial panniculitis. There
are multiple nodular lesions in the inguinal region (A) and lateral flank and dorsal lumbar regions (B). In this cat, lesions progressed slowly over 8 years despite treatment with antimicrobial
drugs. C, Dorsum of an 8-year old dachshund mix with a multidrug resistant Mycobacterium abscessus infection. Multiple draining wounds and extensive scarring are present.
contained mostly neutrophils.76 Heavily vacuolated macrophages
and lipid vacuoles may be present in aspirates from animals with
panniculitis. Organisms may or may not be visible with routine
stains or acid-fast stains. Histopathology shows pyogranulo-
matous inflammation without tubercle formation or necrosis.
Large lipid vacuoles that contain acid-fast bacilli may be present.
Although molecular assays are often used for speciation, direct
use of PCR assays on clinical specimens is often not required for
diagnosis because RGM grow readily on routine culture media
(blood agar). Culture also allows subsequent antimicrobial drug
susceptibility testing to be performed at specialized facilities.
Cytology and histopathology findings for slow-growing
NTM are similar to those for MAC infections. Diagnosis of
slow-growing mycobacterial infection requires specialized
mycobacterial culture or the use of PCR assays. M. xenopi
requires culture at higher temperatures (42°C to 44°C), whereas
other mycobacterial species require lower temperatures (28°C
or 30°C). Organisms such as M. genavense and M. ulcerans can
be difficult to grow on solid media, and so molecular methods
may be required to identify these species.
Treatment and Prognosis
Treatment options for RGM infections include doxycycline, flu-
oroquinolones, clarithromycin, aminoglycosides, sulfonamides,
or carbapenems (see Table 44-4 and Chapter 8). Many cutane-
ous and pulmonary infections respond well to treatment with
doxycycline, aminoglycosides, and/or a fluoroquinolone.74-78
Multidrug-resistant isolates are more difficult to treat; moxi-
floxacin, pradofloxacin, or linezolid could be considered when
other treatment options are not possible.95 As for lepromatous

disease, surgical excision of cutaneous lesions is often followed
by dehiscence and progression if resection is not complete.
However, radical surgical excision by an experienced surgeon
could be considered after antimicrobial drug treatment if lesions
reduce in size but fail to resolve. Intraoperative treatment of
affected cats with an aminoglycoside has been suggested.96
Treatment should continue for 2 months beyond complete reso-
lution of the lesions and is generally required for at least 4 to
12 months.
M. ulcerans infections generally respond to monotherapy
with clarithromycin or a fluoroquinolone.91,92 For animals
with slowly growing mycobacterial infections, options similar
to those used to treat MAC infections are indicated—that is,
combinations of clarithromycin, a rifamycin, and a fluoroqui-
nolone. In human patients, M. kansasii infections are treated
with rifampin and ethambutol, with or without isoniazid.97 The
CASE EXAMPLE
Signalment: “Sarah”, a 5-year-old female spayed husky dog
from Santa Cruz in coastal northern California
History: Sarah was evaluated for a 1-month history of
protrusion of the anus, swelling of the neck, progressive
lethargy, and inappetence. She had been diagnosed with
zinc-responsive dermatitis 3 years prior, and symmetrical
lupoid onychodystrophy 6 months prior. There were two
other dogs and three cats in the household, none of which
were unwell. The dogs lived in a suburban household and
occasionally traveled to southern California, and were
normally fed a commercial dry maintenance dog food.
There was no known history of tick or toxin exposure or
interactions with other wildlife or domestic farm animal
species. The owner was a white non-Hispanic male who
had been sick for some months. His doctor believed he had
acquired the illness from Sarah.
Current Medications: Zinc (2.5 mg/kg q12h PO), tetracycline
(25 mg/kg q12h PO), niacinamide (25 mg/kg q12h PO),
fish oil capsules (1 capsule PO q12h), and vitamin E (20
IU/kg PO q12h) for the zinc-responsive dermatitis and
onychodystrophy. Fipronil with S-methoprene was used for
flea and tick control on a monthly basis.
Physical Examination:
Body Weight: 20 kg
General: Quiet to lethargic, hydrated, responsive, preferred to
lie down but was ambulatory. T=102.9°F (39.4°C), HR = 140
beats/min, RR = 70 breaths/min
Integument: No abnormalities were detected.
Eyes, Ears, Nose, and Throat: A small amount of purulent
ocular discharge was present from the left eye. A fundic
examination was unremarkable. Moderate dental calculus.
Musculoskeletal: Body condition score was 4/9, with
symmetrical muscling. Moderate ventral cervical and
submandibular swelling was present.
Cardiovascular: No murmurs or arrhythmias, femoral pulses
adequate. Mucous membranes were pale pink, with a CRT of 2 s.
CHAPTER 44  Mycobacterial Infections 433
prognosis for disseminated infections in dogs and cats appears
to be guarded to poor.
Public Health Aspects
NTM are opportunistic, environmental mycobacteria that are
not usually transmitted from animals to humans. Disease in
humans resembles that in dogs and cats. Localized cutane-
ous and soft tissue infections with NTM have occurred after
traumatic wounds and lacerations, including after dog and cat
bites.98-100 Infections with RGM can occur after surgical proce-
dures such as liposuction, LASIK surgery, and acupuncture.97
Catheter-related infections have also been described. Dissemi-
nated disease caused by NTM almost always occurs in humans
with HIV infection or other immunosuppressive illness or drug
therapy.
Respiratory: Referred upper airway noises were auscultated.
Gastrointestinal/Genitourinary: The dog’s abdomen
was nonpainful. The rectal mucosa was proliferative
and hyperemic and protruded from the anus. A rectal
examination revealed a nodular diffusely thickened mucosa
with a granular texture and prominent sublumbar lymph
nodes. There was evidence of melena and frank blood on the
glove. No vulvar discharge was noted.
Neurologic: Palpebral, menace, and papillary light responses
were intact bilaterally. No neurologic deficits were appa­rent. A
full neurologic examination was not performed.
Lymph Nodes: Generalized peripheral lymphadenopathy was
present. The mandibular nodes were 5-6 cm in diameter,
lobular and firm. The superficial cervical nodes were 3 cm
in diameter, and the inguinal nodes were enlarged at 2 cm,
with the left larger than the right. The popliteal nodes were
enlarged at 2-3 cm bilaterally.
Laboratory Findings:
CBC:
HCT 22% (40%-55%)
MCV 52 fL (65-75 fL)
MCHC 30 g/dL (33-36 g/dL)
Reticulocyte count 25,200 cells/µL
WBC 40,510 cells/µL (6000-13,000 cells/µL)
Neutrophils 29,977 cells/µL (3000-10,500 cells/µL)
Band neutrophils 2431 cells/µL
Lymphocytes 2026 cells/µL (1000-4000 cells/µL)
Monocytes 2431 cells/µL (150-1200 cells/µL)
Eosinophils 3646 cells/µL (0-1500 cells/µL)
Platelets 387,000 platelets/µL (150,000-400,000 platelets/µL).
Band and mature neutrophils were slightly toxic.
Serum Chemistry Profile:
Sodium 132 mmol/L (145-154 mmol/L)
Potassium 3.5 mmol/L (3.6-5.3 mmol/L)
Chloride 103 mmol/L (108-118 mmol/L)
Bicarbonate 17 mmol/L (16-26 mmol/L)
Phosphorus 4.1 mg/dL (3.0-6.2 mg/dL)
Calcium 8.6 mg/dL (9.7-11.5 mg/dl)
BUN 6 mg/dL (5-21 mg/dL)
Continued
434 SECTION 2  Bacterial Diseases
Creatinine 0.6 mg/dL (0.3-1.2 mg/dL)
Glucose 93 mg/dL (64-123 mg/dL)
Total protein 4.3 g/dL (5.4-7.6 g/dL)
Albumin 2.1 g/dL (3.0-4.4 g/dL)
Globulin 2.2 g/dL (1.8-3.9 g/dL)
ALT 43 U/L (19-67 U/L)
AST 51 U/L (19-42 U/L)
ALP 115 U/L (21-170 U/L)
Creatine kinase 202 U/L (51-399 U/L)
GGT < 3 U/L (0-6 U/L)
Cholesterol 146 mg/dL (135-361 mg/dL)
Total bilirubin 0 mg/dL (0-0.2 mg/dL)
Magnesium 2.0 mg/dL (1.5-2.6 mg/dL).
Urinalysis: SGr 1.005; pH 7.5; no protein, bilirubin, hemoprotein,
or glucose; rare WBC/HPF, 0 RBC/HPF, few lipid droplets.
Imaging Findings:
Thoracic Radiographs: No abnormalities were detected.
Abdominal Ultrasound: All abdominal lymph nodes
were enlarged and irregular. There were focal regions of
thickening and irregularity within the small bowel. The liver
and spleen were unremarkable.
Aspiration Cytology (Mandibular Lymph Node):
Multiple, variably sized aggregates of macrophages were
scattered along with numerous karyolytic and degenerated
neutrophils and a residual lymphoid population in a light pink
background that contained low numbers of erythrocytes
and lysed cells. The macrophages were large and distended
with intracellular, negatively staining thick, relatively short,
rod-shaped bacteria. Intracellular organisms were noted
within neutrophils and large multinucleated giant cells, as
well as being located extracellularly. The bacilli were acid
fast. Interpretation: Pyogranulomatous inflammation due to
infection with Mycobacterium spp.
Microbiologic Testing: Real-time PCR assay for Mycobacter­ium
genus organisms (lymph node aspirate): Positive
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96. O’Brien CR, Fyfe JA, Malik R. Infections caused by rapidly-­
growing mycobacteria. In: Greene CE, ed. Infectious Diseases of
the Dog and Cat. 4th ed. St. Louis, MO: Elsevier Saunders; 2012:
515-521.
97. Brown-Elliott BA, Wallace RJ. Infections due to non-tuberculous
mycobacteria other than Mycobacterium avium-intracellulare. In:
Mandell GL, Bennett JE, Dolin R, eds. Principles and Practice of
Infectious Diseases. Philadelphia, PA: Elsevier; 2010:3191-3198.
98. Southern Jr PM. Tenosynovitis caused by Mycobacterium kansasii
associated with a dog bite. Am J Med Sci. 2004;327:258-261.
99. Ariel I, Haas H, Weinberg H, et  al. Mycobacterium fortuitum
granulomatous synovitis caused by a dog bite. J Hand Surg Am.
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100. Ngan N, Morris A, de Chalain T. Mycobacterium fortuitum
infection caused by a cat bite. N Z Med J. 2005;118:U1354.
101. Rodwell TC, Kapasi AJ, Moore M, et al. Tracing the origins of
Mycobacterium bovis tuberculosis in humans in the USA to cattle
in Mexico using spoligotyping. Int J Infect Dis. 2010;14(suppl 3):
e129-e135.
CHAPTER 45
Salmonellosis
Jane E. Sykes and Stanley L. Marks
Overview of Salmonellosis
First Described: Washington, DC, United States, 1885 (Theo-
bald Smith and Daniel Salmon)1
Cause: Salmonella spp. (gram-negative rods that belong to
the Enterobacteriaceae)
Affected Hosts: A large variety of warm-blooded animals
and humans; cold-blooded animals may be subclinically
infected
Geographic Distribution: Worldwide
Primary Mode of Transmission: Fecal-oral
Major Clinical Signs: Fever, lethargy, anorexia, diarrhea, vom-
iting, and less commonly reproductive failure, neurologic
and/or respiratory signs
Differential Diagnoses: Differential diagnoses for suspected
Salmonella enterocolitis include canine and feline parvo-
virus infection, canine distemper virus infection, campylo-
bacteriosis, clostridial diarrhea, salmon poisoning disease,
giardiasis, tritrichomoniasis (cats), cryptosporidiosis, whip-
worms, leptospirosis, dietary indiscretion, gastrointestinal
foreign body, pancreatitis, inflammatory bowel disease,
lymphoma, hypoadrenocorticism, hyperthyroidism, toxins
(including drugs)
Human Health Significance: Important zoonosis. Dogs and
cats may be a source of human infection with some Sal-
monella serotypes, some of which may be resistant to
multiple antimicrobial drugs.
Etiology and Epidemiology
Salmonella are gram-negative, motile, non–spore-forming fac-
ultative anaerobic rods that belong to the family Enterobacte-
riaceae. Most Salmonella exist in two phases, which differ in
composition of their flagellar antigens.
Salmonella are ubiquitous organisms that can be isolated
from the intestinal tracts of an enormous variety of animal spe-
cies and are a major cause of enterocolitis and sometimes severe
systemic illness in humans and animals. Infections can be trans-
ferred between animal species and are zoonotic. Many infec-
tions are food borne, especially in association with milk, meat,
and eggs. Salmonella spp. have a remarkable ability to survive
long periods of time (weeks to years) in the environment, and
so fomite transmission is important. In addition, Salmonella can
multiply rapidly in contaminated food left at room temperature
and can survive freezing for several weeks.2
The genus Salmonella comprises two species, Salmonella
enterica and Salmonella bongori, each of which contains
multiple serotypes.3 Serotypes of S. bongori are generally asso-
ciated with cold-blooded animals and rarely have been isolated
from warm-blooded animals, including a dog with diarrhea.4
There are six subspecies of S. enterica (Table 45-1) and approx-
imately 2500 different serotypes. These are identified on the
basis of agglutination reactions of their O (somatic) and H
(flagellar) antigens (see Chapter 36). Subspecies I serotypes,
which are responsible for almost all disease in humans and
warm-blooded animals worldwide, are assigned names such
as S. ser. Typhimurium. Unless named before 1966, serotypes
that belong to other subspecies are designated using antigenic
formulas. The formulas describe the subspecies, O antigens: H
antigens (phase 1): and if present, H antigens (phase 2) (e.g., S.
IV 45:g,z51:—).
Certain serotypes of Salmonella are host restricted, whereas
others infect a broad range of host species. Salmonella serotype
Typhi, for example, is adapted to humans, causing typhoid
fever, and does not infect animals. Other serotypes are adapted
to certain host species, with rare transmission to other hosts.
Salmonella serotype Typhimurium is the most commonly iso-
lated serotype from diseased humans and animals.
Infection of dogs and cats with Salmonella has been associ-
ated with the feeding of raw meat diets,5-9 although commercial
dry and raw dog food and pig ear pet treats have also become
contaminated with the organism.5,10-13 Occasionally, co-infec-
tions with multiple Salmonella serotypes occur.14 Outbreaks of
S. ser. Typhimurium infection in cats have been associated with
seasonal bird migrations (“songbird fever”).
Salmonella is generally isolated less than 1% of the time from
the feces of dogs and cats that are fed processed commercial
TABLE 45-1
Classification of Salmonellae
Salmonella Salmonella
Species ­Subspecies Example Serotypes
Salmonella enterica (I) Salmonella ser. Typhimurium
enterica S. ser. Typhi
S. ser. Choleraesuis
salamae (II) S. ser. Greenside
arizonae (IIIa) S. IIIa 18:z4,z23:—
diarizonae (IIIb) S. IIIb 60:k:z
houtenae (IV) S. ser. Marina
indica (VI) S. ser. Srinagar
Salmonella V S. ser. Brookfield
bongori
437
438 SECTION 2  Bacterial Diseases
pet foods,15-19 although one study of household pet dogs in
Ontario, Canada revealed a shedding prevalence of 23%.20
The highest rates of infection have been found in group-housed
dogs, especially those fed raw meat diets, such as racing sled
dogs and greyhound breeding facilities, where prevalences have
exceeded 75%.8,21 Salmonella was isolated from the feces of 18
of 26 (69%) healthy pre-race Alaskan sled dogs, and 19 of 30
(63%) diarrheic racing Alaskan sled dogs, which underscored
the lack of an association between the isolation of Salmonella
and clinical diarrhea.8 Coprophagic behavior may also contrib-
ute to salmonellosis in dogs. In cats, the feeding of raw poultry
may increase risk for Salmonella infection,22 and occasionally,
high prevalences of infection have been detected in group-
housed cats18 and shelter cats. Ingestion of infected wild bird
species by cats during seasonal bird migrations can also lead
to salmonellosis (songbird fever). In a study of diarrheic and
nondiarrheic shelter cats in Florida, the prevalence of Salmo-
nella shedding as determined by PCR assay was 6% and 4%,
respectively.23 Susceptibility to shedding and disease is highest
in immunosuppressed dogs and cats, including young animals,
pregnant animals, those in overcrowded conditions, and those
with underlying immunosuppressive illness (such as neoplasia,
diabetes mellitus, retroviral infection, and immune-mediated
disease) or immunosuppressive drug therapy.24,25
Clinical Features
Signs and Their Pathogenesis
After ingestion, salmonellae that survive the acidic environment
of the stomach gain access to the intestine, where they disrupt
tight junctions and actively invade (1) enterocytes; (2) M cells
within the ileum (which sample luminal antigens); and (3) den-
dritic cells, which send protrusions into the gut lumen between
intestinal epithelial cells. Interaction of Salmonella with immune
cells and epithelial cells within the gut leads to production of
cytokines and chemokines, with massive influx of lymphocytes,
macrophages, and neutrophils, sometimes with severe epithe-
lial injury and mucosal sloughing.26 Localization in the mesen-
teric lymph nodes, Peyer’s patches, solitary intestinal lymphoid
FIGURE 45-1  Large, chronic pancreatic abscess due to Salmonella Typhimurium associated with a pancreatic adenocarcinoma in a 15-year-old castrated male domestic shorthair
cat. The mass was multicystic and fluctuant, and when cut open was filled with cloudy fluid and lined by thick tan debris. (Courtesy University of California, Davis, Veterinary Anatomic
­Pathology Service.)
tissues, and intestinal epithelium, along with evasion of the host
immune response, is followed by persistent shedding, often for
several weeks, after which animals can become latently infected.
Reactivation of shedding, with or without clinical illness, may
occur, sometimes as a result of stress or immunosuppression.27,28
If the organism has properties that allow it to spread systemi-
cally and host defenses are impaired, bacteremia and extraint-
estinal infection follows.29 More virulent strains have a greater
ability to multiply intracellularly in nonphagocytic cells. Fever,
hypoglycemia, and leukopenia occur. The Salmonella lipo-
polysaccharide (LPS) can then induce endotoxic shock, with
hypotension and activation of the complement and coagulation
cascade. The coagulation cascade is also activated with exposure
to Salmonella porins, which are hydrophobic outer membrane
proteins. Disseminated intravascular coagulation (DIC) follows.
Dogs and cats infected with Salmonella spp. may show no signs
or they may develop enterocolitis, focal suppurative infection, or
severe systemic illness. The majority of dogs are chronically and
subclinically infected. When disease occurs, signs often begin 3 to
5 days after infection or onset of immunosuppression. Fever (occa-
sionally as high as 106°F [41°C]), lethargy, and anorexia may be
followed by abdominal pain, vomiting, and watery to mucoid,
often hemorrhagic diarrhea, and dehydration. Weight loss may be
seen. Diarrhea may take several weeks to resolve. Rarely, chronic
intermittent diarrhea that lasts up to 8 weeks develops.30
Severely affected animals develop signs of septic shock (see
Chapter 86). Neurologic signs, and signs that relate to endocar-
ditis, arthritis, pancreatitis, pneumonia, peritonitis, and chole-
cystitis occur in some animals. Infected cats may develop severe
enterocolitis, with fever, pancytopenia, and hyperbilirubinemia.
Some cats show persistent fever and anorexia with no diarrhea.
Conjunctivitis has been described in cats in association with Sal-
monella infection.31
Occasionally salmonellae localize in a particular organ, such
as the lungs or urinary tract (Figure 45-1). Signs of dysfunction
of that organ system may follow, even when enteric signs or
positive fecal culture results are absent.32 Abortion and stillbirth
can occur after transplacental infection, and the dam may also
infect her neonates, which leads to fading puppies.33-35

Diagnosis
The traditional diagnosis of canine and feline salmonellosis is
made based on culture of the organism in conjunction with
clinical signs and assessment of potential risk factors, such as
hospitalization, age, environmental exposure, and antibiotic
administration. Diagnostic assays available for salmonellosis in
dogs and cats are shown in Table 45-2.
Laboratory Abnormalities
Complete Blood Count
The CBC in dogs or cats with salmonellosis may be unremark-
able or may show a neutrophilia with a left shift; in bacteremic
animals, anemia, a degenerative left shift, toxic neutrophils,
lymphopenia, and thrombocytopenia are usually present.
Rarely, intracellular bacteria are seen within neutrophils.
Serum Chemistry Profile
In animals with severe salmonellosis, changes on the serum
chemistry profile include elevated liver enzyme activities, hyper-
bilirubinemia, azotemia, hypoalbuminemia, hypocholesterol-
emia, hypoglycemia, and electrolyte abnormalities.
Urinalysis
Salmonella spp. occasionally localize in the kidneys, with asso-
ciated isosthenuria, pyuria, proteinuria, casts, and bacteriuria.
Clotting Function
Dogs and cats that develop DIC may have clotting func-
tion abnormalities that include prolonged coagulation times,
increased fibrin degradation product concentrations, and
decreased antithrombin concentrations.
Diagnostic Imaging
Plain Radiography
Thoracic radiographs in animals with salmonellosis may reveal
bronchointerstitial to alveolar infiltrates in animals with pneu-
monia, or pleural effusion with pyothorax. Abdominal radio-
graphs can reveal fluid-filled intestines, decreased serosal detail,
and/or mild hepatomegaly.
Sonographic Findings
Depending on the severity of the infection, abdominal
ultrasound may be unremarkable in dogs and cats with
salmonellosis, or show changes that include abdominal
TABLE 45-2
Diagnostic Assays Available for Salmonellosis in Dogs and Cats
Assay Specimen Type Target
Bacterial Feces, blood, synovial fluid, tis- Salmonella species
isolation sue aspirates, bronchoalveolar
­lavage fluid, peritoneal or pleu-
ral effusions, urine, CSF, tissues
obtained at necropsy
PCR As for isolation Salmonella DNA
CHAPTER 45  Salmonellosis 439
lymphadenomegaly, intestinal wall thickening, fluid-filled bowel
segments, ­hepatomegaly, splenomegaly, or ascites.
Microbiologic Testing
Bacterial Isolation
A diagnosis of salmonellosis can be made when Salmonella is
isolated from a normally sterile site, such as the blood, bron-
choalveolar lavage specimens, synovial fluid, or urine specimens
collected by cystocentesis. Isolation of Salmonella from feces
does not confirm that the organism is the cause of disease, but
it can raise suspicion that Salmonella may be playing a role in
enterocolitis, and it has zoonotic significance (see Public Health
Aspects, later). Isolation also allows antimicrobial susceptibility
testing, which is important because many isolates of Salmonella
are resistant to multiple antimicrobial drugs.
Salmonella grow readily at 37°C on routine bacteriologic
media from specimens that have been collected from a nor-
mally sterile site. Selective media are required for specimens
that are heavily contaminated with other bacteria such as feces.
Examples of selective media include enrichment broths such
as tetrathionate broth or Selenite F broth. After enrichment,
subculturing is performed on a medium such as deoxycho-
late, which also favors growth of Salmonella. Many veteri-
nary laboratories incorporate such protocols for isolation of
Salmonella as part of a panel for detection of enteropatho-
genic bacteria. Once Salmonella is isolated, the organism can
be identified based on biochemical reactions and serotyping,
which involves agglutination testing with O and H antisera.
In the future, use of whole cell matrix-assisted laser desorp-
tion ionization-time-of-flight (MALDI-TOF) mass spectrom-
etry in veterinary clinical microbiology laboratories should
allow identification of Salmonella at the species and subspe-
cies level within minutes.36 It also can identify some important
S. enterica subsp. enterica to the serovar level, and could
reduce the need for laborious subtyping procedures.37
Molecular Diagnosis Using the Polymerase Chain Reaction
Some veterinary diagnostic laboratories offer PCR assays for
Salmonella, some of which have been shown as highly sensi-
tive and specific when compared with culture.38,39 PCR is more
rapid than culture but currently does not provide information
on antimicrobial susceptibility. It has been recommended that
PCR assay after overnight enrichment in a nonselective broth be
adopted as the gold standard for diagnosis, and that all speci-
mens that test positive by PCR assay be cultured using selective
Performance
Sensitive and specific. False negatives may occur, especially
following antimicrobial therapy. Isolation from feces
does not imply that Salmonella is the cause of disease.
Allows subsequent antimicrobial susceptibility testing.
Rapid (within hours); can be highly sensitive and specific.
Positive results from feces do not imply that Salmonella
is the cause of disease. Does not allow susceptibility test-
ing or typing for epidemiologic purposes.
440 SECTION 2  Bacterial Diseases
enrichment to isolate and identify the infecting organism.40 As
with culture, detection of the organism in feces using PCR assays
does not prove that it is the cause of gastrointestinal illness.
Pathologic Findings
Gross Pathologic Findings
Gross necropsy findings in dogs and cats with severe salmo-
nellosis include widespread petechial and ecchymotic hemor-
rhages, hemorrhagic enteritis, abscesses within parenchymal
organs, enlarged mesenteric lymph nodes, and fibrinohemor-
rhagic ascites fluid. Pulmonary consolidation and edema may
also be present.
Histopathologic Findings
Histopathologic lesions in dogs and cats with salmonellosis
are highly variable. Changes in severely affected animals may
include suppurative pneumonia, necrotizing hepatitis, and nec-
rotizing and fibrinohemorrhagic enterocolitis, typhlitis, and
cholecystitis. More chronic lesions such as chronic cholecys-
titis have also been described.41,42 Sometimes, gram-negative
bacilli are identified within lesions with tissue Gram stains
(Figure 45-2).
Treatment and Prognosis
The movement of dogs and cats that test positive for Salmonella
should be limited, and if possible, hospitalized animals should
be isolated. The mere detection of Salmonella in the feces of
dogs and cats with uncomplicated diarrhea does not warrant
antimicrobial administration, and supportive care only is rec-
ommended. Most of these animals have self-limiting disease and
shedding, and injudicious antimicrobial administration has the
potential to prolong the carrier state and contribute to antimi-
crobial resistance. Treatment of an animal with uncomplicated
diarrhea is also not advocated if the owner is immunocompro-
mised, although appropriate husbandry recommendations and
barrier control must be enforced.
FIGURE 45-2  Gram-negative bacilli (arrow) in the lungs of a cat with suppurative
cholecystitis and pneumonia due to Salmonella arizonae. The cat had been treated with
prednisone and cyclosporine for immune-mediated hemolytic anemia. Brown and Benn
stain, 1000× magnification.
Dogs and cats with systemic salmonellosis may require
aggressive intravenous fluid therapy and colloidal support with
hydroxyethyl starch or plasma, together with parenteral anti-
microbial drugs. The choice of antimicrobial drugs should be
based on the results of culture and susceptibility, because of the
potential for resistance to multiple antimicrobial drugs through
plasmid transfer. In the event of systemic disease, administra-
tion of a combination of ampicillin and a fluoroquinolone is
advocated as empiric therapy while awaiting results of culture
and susceptibility. Because of the importance of endotoxin in
the pathogenesis of sepsis due to gram-negative bacteria such
as Salmonella, considerable effort has been invested in devel-
opment and application of drugs targeting endotoxin.43 Clear
benefit through reduction in mortality has not yet been dem-
onstrated for these treatments, and to date they have not been
widely used in dogs and cats. Most recently, investigations have
been focused on antagonists of Toll-like receptor 4, which is the
LPS receptor.44 Eritoran tetrasodium (E5564) and TAK-242 are
examples of TLR4 antagonists that have been used in clinical
trials involving septic humans.45
Immunity and Vaccination
Salmonella spp. can persist in the host by interfering with den-
dritic cell function in the intestinal tract. The organism pene-
trates dendritic cells and avoids lysosomal degradation, which
prevents subsequent antigen presentation by dendritic cells on
molecules of the major histocompatibility complex.46 Reduced
intracellular proliferation of the organism within antigen-
presenting cells may also limit antigen presentation and the
development of an effective immune response.47 Although Sal-
monella vaccines have been developed, none are available for
dogs and cats.
Prevention of Enteric Bacterial Infections
Prevention of enteric bacterial infections includes feeding dogs
and cats properly cooked foods, hand washing before and after
handling pets and pet food, and prevention of coprophagic
behavior. Proper handling and storage of pet foods can also
limit the potential for food contamination during storage by
rodents, insects, and human hands.
Within the hospital setting, fomites such as food dishes,
endoscopes, proctoscopes, and rectal thermometers should be
disinfected between uses, and single-use disposable thermom-
eter covers should be used. Cages should be cleaned and prop-
erly disinfected (see Chapter 11) between animals. Blood donors
should not be housed with the regular hospital population, as
they may be a source of Salmonella infection.
Dogs and cats diagnosed with salmonellosis or Salmonella
shedding should be isolated from other animals until shedding
stops. Fecal cultures should be repeated every 2 weeks, and ter-
mination of shedding is identified when three successive nega-
tive cultures have been obtained.
Public Health Aspects
All enteric bacterial infections of dogs and cats have the poten-
tial to infect and cause illness in humans. Nontyphoid salmo-
nellosis (salmonellosis other than that caused by S. ser. Typhi)
can be transmitted from domestic animals to humans and
cause serious enterocolitis, with fever, severe abdominal pain,

diarrhea, nausea, inappetence, chills, and headache. Serious
complications tend to occur in children, the elderly, or other-
wise immunocompromised individuals such as those infected
with HIV. The majority of cases result from ingestion of
improperly cooked foods of animal origin, such as meat, milk,
and eggs. Most, if not all, pet reptiles carry Salmonella, and
there has been considerable effort to educate the public about
the hazards of reptile-associated salmonellosis. Dogs, and to a
lesser extent cats, are less commonly incriminated as a source of
Salmonella. Of particular concern, multidrug-resistant Salmo-
nella strains, such as S. ser. Typhimurium strain DT 104, have
CASE EXAMPLE
FIGURE 45-3  “Sampson”, a 3-year-old male castrated shepherd mix with
salmonellosis.
Signalment: “Sampson,” a 3-year-old male castrated shepherd
mix from Fresno in southern California (Figure 45-3).
History: Sampson was brought to a veterinarian for evaluation
of a 3-month history of bilateral tarsal joint swelling. Some
clinical improvement had been noted after treatment with
oral tetracycline (28 mg/kg PO q8h) for 2 weeks. Subsequently,
he was treated with oral prednisone (1.1 mg/kg PO q12h). Two
weeks after the prednisone treatment was initiated, Sampson
became nonambulatory in his pelvic limbs, and all of his
peripheral joints became swollen and he was referred to the
University of California, Davis, Veterinary Medical Teaching
Hospital. A mass had been removed from his left dorsolateral
CHAPTER 45  Salmonellosis 441
been isolated from dogs, cats, and reptiles.20,48 People exposed
to dogs and cats that are fed raw meat diets are at increased
risk of exposure.
Proper hand washing after handling pets, pet food, and fomi-
tes such as bedding and food dishes can help prevent zoonotic
transmission of Salmonella. Reptile ownership by families with
children younger than 5 years of age or among the immuno-
suppressed is discouraged by public health authorities.49 Proper
food handling practices, including separating raw meat from
vegetables and proper cooking of meat, can also reduce salmo-
nellosis in humans.
thorax 3 weeks before referral that was assumed to be a foxtail
abscess. Sampson had a good appetite. Increased thirst and
urination had been noted after the prednisone treatment was
initiated. There had been no coughing, sneezing, vomiting, or
diarrhea. He was fed a duck- and potato-based diet for food
allergy dermatitis and had been treated intermittently with
prednisone, 4 mg PO q24h, for this condition since he was 6
months old. He had not traveled outside his local area, but
he lived on a ranch and was known to consume horse feces
and dead wild animals. There was no known tick exposure. He
was up to date on vaccinations including canine distemper,
adenovirus, parvovirus, and rabies vaccines.
Current medications: Prednisone 0.9 mg/kg PO q12h
Physical Examination:
Body Weight: 35.8 kg
General: Quiet, alert and responsive, 5% to 7% dehydrated.
Recumbent. T = 102.9°F (39.4°C), HR = 140 beats/min,
panting, mucous membranes pink and tacky, CRT = 1 s.
Integument: Multifocal epidermal collarettes were present
and the haircoat was dry. A shaved area was present on the
left lateral thoracic wall.
Eyes, Ears, Nose, and Throat: Moderate periodontal disease was
present. A fundic examination was within normal limits.
Musculoskeletal: Body condition score was 5/9 with pelvic
limb muscle atrophy. Pain and effusion of the carpi, tarsi,
and stifles with decreased range of motion was noted.
Spinal and pericervical pain was detected on palpation of
these regions.
Cardiovascular: Weak but synchronous femoral pulses were
noted. No murmurs or arrhythmias were auscultated.
Respiratory: There were moderately increased breath sounds
in all lung fields.
Gastrointestinal/Genitourinary: The abdomen was tense
on palpation and moderate hepatomegaly was detected.
The dog urinated a large amount of odiferous urine during
the examination. No abnormalities were detected on rectal
examination.
Lymph Nodes: Moderate peripheral lymphadenopathy was
present. Mandibular, superficial cervical, and popliteal
lymph nodes were all 1.5 to 2.5 cm in diameter.
Laboratory Findings:
CBC:
HCT 35.4% (40%-55%)
MCV 67 fL (65-75 fL)
MCHC 35.6 g/dL (33-36 g/dL)
Continued
442 SECTION 2  Bacterial Diseases

Reticulocytes 101,200 cells/µL (7000-65,000 cells/µL)

WBC 7870 cells/µL (6000-13,000 cells/µL)
Neutrophils 6139 cells/µL (3000-10,500 cells/µL)
Band neutrophils 1495 cells/µL
Metamyelocytes 79 cells/µL
Lymphocytes 0 cells/µL (1000-4000 cells/µL)
Monocytes 79 cells/µL (150-1200 cells/µL)
Platelets 419,000 platelets/µL (150,000-400,000 platelets/µL).
Neutrophils, band neutrophils, and metamyelocytes all
showed moderate toxic changes.
Serum Chemistry Profile:
Sodium 148 mmol/L (145-154 mmol/L)
Potassium 4.4 mmol/L (3.6-5.3 mmol/L)
Chloride 105 mmol/L (108-118 mmol/L)
Bicarbonate 14 mmol/L (16-26 mmol/L)
Phosphorus 5.8 mg/dL (3.0-6.2 mg/dL)
Calcium 10.3 mg/dL (9.7-11.5 mg/dl)
BUN 10 mg/dL (5-21 mg/dL)
Creatinine 0.4 mg/dL (0.3-1.2 mg/dL)
Glucose 111 mg/dL (64-123 mg/dL)
FIGURE 45-4  Thickening and hyperechogenicity of the posterior tricuspid valve
Total protein 6.1 g/dL (5.4-7.6 g/dL)
­leaflet (arrowhead) in a dog with Salmonella arizonae bacteremia.
Albumin 2.3 g/dL (3.0-4.4 g/dL)
globulin 3.8 g/dL (1.8-3.9 g/dL)
ALT 560 U/L (19-67 U/L) IFA for antibodies to Bartonella vinsonii subspecies berkhoffii
AST 72 U/L (19-42 U/L) was negative. Serology for Coccidioides spp. antibodies was
ALP 3318 U/L (21-170 U/L) also negative.
Creatine kinase 197 U/L (51-399 U/L) Aerobic bacterial culture of blood, joint fluid, and urine collect-
Gamma GT 59 U/L (0-6 U/L) ed by cystocentesis: Salmonella arizonae, susceptible to all
Cholesterol 212 mg/dL (135-361 mg/dL) antimicrobials tested.
Total bilirubin 0.3 mg/dL (0-0.2 mg/dL). Diagnosis: Salmonella arizonae septic polyarthritis,
Urinalysis: SGr 1.010; pH 8.0, 1+ protein (SSA), 3+ hemoprotein, bacteremia, and possible tricuspid valve endocarditis.
no bilirubin or glucose, 25-35 WBC/HPF, 10-15 RBC/HPF, Treatment: Before obtaining susceptibility results, Sampson
many rods. was treated with intravenous crystalloids, opioids for pain
Imaging Findings: control, enrofloxacin (5 mg/kg IV q24h), and ampicillin (30
Thoracic Radiographs: The cardiovascular and pulmonary mg/kg IV q8h). The ampicillin was discontinued when the
structures appeared within normal limits. susceptibility results became available. The prednisone was
Right and Left Carpal, Stifle, and Tarsal Radiographs: tapered over a 3-day period and discontinued. The dog was
Intracapsular soft tissue swelling was associated with all moved to an isolation ward on diagnosis of salmonellosis.
joints. During hospitalization, Sampson’s appetite declined initially,
Spinal Radiographs: No abnormalities were detected. and he remained very painful for 6 days. After that time, he
Abdominal Ultrasound: The liver was enlarged and was able to sit up, began eating, and was discharged 8 days
hyperechoic. There was echogenic sediment in the urinary after admission.
bladder. Comments: Salmonella arizonae infections in humans have
Echocardiogram: Thickening and hyperechogenicity of the been associated with direct or indirect contact with reptiles.
posterior leaflet of the tricuspid valve was noted (arrowhead) Reptile-associated salmonellosis is an increasing problem
which raised suspicion for endocarditis (Figure 45-4). in humans in the United States and is especially a problem
Cytology Findings: Arthrocentesis of right and left carpi, in infants and immunocompromised adults in contact with
right tarsus and right stifle: Purulent inflammation was snakes and iguanas, or those who eat sun-dried rattlesnake
noted in the right tarsus, right stifle, and left carpus, with meat for medicinal purposes. Horse feces from the farm were
approximately 85% neutrophils and the remaining cells cultured for Salmonella and were negative. It is possible that
were a mixture of small and large mononuclear cells. In the infection in this dog occurred as a result of ingestion
the right stifle, degenerate neutrophils are visualized, and of wild reptiles. The long history of glucocorticoid therapy
some contained intracellular rod-shaped bacteria. for atopic dermatitis in this dog may have contributed
Microbiologic Testing: Serologic testing: An in-clinic to immunosuppression and predisposition to systemic
ELISA for antibodies to Borrelia burgdorferi, Anaplasma salmonellosis.
phagocytophilum, and Ehrlichia canis was negative. Serum

SUGGESTED READINGS
Behravesh CB, Ferraro A, Deasy M, et al. Human Salmonella infections
linked to contaminated dry dog and cat food, 2006-2008. Pediatrics.
2010;126(3):477-483.
Grassl GA, Finlay BB. Pathogenesis of enteric Salmonella infections.
Curr Opin Gastroenterol. 2008;24(1):22-26.
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CHAPTER 46
Enteric Escherichia coli Infections
Jane E. Sykes and Stanley L. Marks
Overview of Enteric Escherichia coli Infections
First Described: Germany, 1885 (Theodor Escherich)1
Cause: Escherichia coli (gram-negative rods that belong to the
Enterobacteriaceae)
Affected Hosts: Humans and a large variety of animals
Geographic Distribution: Worldwide
Primary Mode of Transmission: Fecal-oral
Major Clinical Signs: Fever, lethargy, inappetence, diarrhea,
vomiting
Differential Diagnoses: Differential diagnoses for suspected
E. coli ­enterocolitis include canine and feline parvovi-
rus infection, canine distemper virus infection, salmon
poisoning disease, campylobacteriosis, clostridial
diarrhea, salmonellosis, giardiasis, tritrichomoniasis
(cats), cryptosporidiosis, whipworms, pythiosis, histo-
plasmosis, leptospirosis, dietary indiscretion, gastroin-
testinal foreign body, pancreatitis, inflammatory bowel
disease, lymphoma, hypoadrenocorticism, hyperthyroid-
ism (cats), toxins (including drugs).
Human Health Significance: Dogs and cats may be a source of
human infection with some E. coli serotypes.
Etiology and Epidemiology
Escherichia coli are pleomorphic gram-negative, non–spore-
forming rods that belong to the family Enterobacteriaceae. Like
Salmonella, E. coli can survive long periods of time in feces,
dust, and water. They are part of the normal flora of the gas-
trointestinal tract but can be associated with enterocolitis in the
presence of bacterial virulence factors and impaired local or
systemic immunity. As with Salmonella infections, E. coli infec-
tions can be transferred between animal species and some may
be zoonotic; many infections are food borne.
There are more than 170 serogroups of E. coli based on the
identity of the bacterial O (somatic) antigen, the sugar that is
on the most external portion of the bacterial lipopolysaccha-
ride (LPS) (see Chapter 36). Organisms are also identified on
the basis of their H (flagellar) antigens—for example, O157:H7
E. coli. Both the bacterial LPS (endotoxin) and the flagellar anti-
gens are bacterial virulence factors. Other virulence factors in
E. coli include adhesins, bacterial exotoxins such as cytotoxic
necrotizing factor (CNF), hemolysins, heat-labile (LT) and heat-
stable (ST) enterotoxins, and the capsular polysaccharide (K).
E. coli strains that cause gastrointestinal disease have been
divided into seven distinct pathogenic categories (pathovars).
Each pathovar is defined by a characteristic set of virulence fac-
tors that act in concert to determine the clinical, pathologic, and
epidemiologic features of the disease they cause. The seven path-
ovars include enteropathogenic E. coli (EPEC), enterotoxigenic
E. coli (ETEC), enterohemorrhagic E. coli (EHEC), necrotoxi-
genic E. coli (NTEC), enteroinvasive E. coli (EIEC), enteroag-
gregative E. coli (EAEC), and adherent-invasive E. coli (AIEC)
(Table 46-1). Currently, the epidemiology of many of these strains
and their role in disease causation is not well defined in small
animals, with most work having been done in humans and farm
animal species. Many strains have been isolated from both nondi-
arrheic and diarrheic dogs and cats. An association between EPEC
and EHEC (including H7:O157) infection and diarrhea in dogs
has been detected.2,3 In addition, AIEC strains have been well
associated with granulomatous colitis in boxer dogs.4
Clinical Features
Signs and Their Pathogenesis
EPEC strains carry the eaeA gene on their chromosome (E. coli
attaching effacing), which is located in a “pathogenicity island”
referred to as LEE (locus of enterocyte effacement). The eaeA
gene encodes a 94-kDa protein, intimin, which allows the organ-
ism to adhere intimately to intestinal epithelial cells. This leads
to local effacement of the microvilli and formation of numerous,
actin-rich pedestals on which the bacteria reside (Figure 46-1).5
The bacteria secrete their own receptor, the Tir receptor, and
Tir-intimin interactions are involved in pedestal formation.
The pedestals may help the bacteria to remain extracellular,
and thus escape immune recognition by the host. The result-
ing characteristic histopathologic lesions on enterocytes have
been referred to as “attaching and effacing lesions.” Subsequent
signal transduction events induced by EPEC may be associated
with loss of tight junction integrity and altered electrolyte trans-
port, with associated watery diarrhea. The EPEC pathovar does
not produce Shiga toxin, Shiga-like toxin, or verotoxins. EPEC
serotypes that have been associated with diarrhea in humans
have been identified in dogs and cats.6,7
ETEC are a major cause of diarrhea in human infants in
developing countries and are the agents most frequently respon-
sive for traveler’s diarrhea. ETEC pathovars have been associ-
ated with up to 31% of cases of canine diarrhea, particularly
in young dogs.8-10 Most of the canine ETEC strains express ST
enterotoxin, whereas ETEC strains that produce LT enterotoxin
are rarely found. The bacteria adhere to the proximal small
intestinal mucosa and produce plasmid-encoded LT and ST
enterotoxins. LT is related to cholera toxin and is taken up by
enterocytes. Once within intestinal epithelial cells, it activates
adenylate cyclase, which leads to increased concentrations of
intracellular cyclic AMP with resultant secretion of electrolytes
445
446 SECTION 2  Bacterial Diseases

TABLE 46-1
Pathogenic Strains of Escherichia coli That Cause Enterocolitis
Strain Lesion Signs Virulence Characteristic
Enteropathogenic E. coli Attaching and effacing lesions Watery diarrhea eaeA gene (encodes
(EPEC) (effacement of microvilli and intimin)
pedestal formation)
Enterotoxigenic E. coli Adhere using fimbria and Diarrhea LT and ST production
(ETEC) produce heat-labile (LT)
and/or heat-stable (ST) toxins
Enterohemorrhagic E. coli Produce Shiga-like toxins Diarrhea, hemorrhagic colitis, Shiga-like toxin
(EHEC); also known as ­(verotoxins) that cause hemolytic-uremic syndrome ­production, see also
Shiga-toxin–producing vascular endothelial damage, EPEC
E. coli (STEC) or verotoxin- also produce attaching and
producing E. coli (VTEC) effacing lesions
Necrotoxigenic E. coli Produce cytotoxic necrotizing Diarrhea, bacteremia, urinary CNFs
(NTEC) ­factors (CNFs) tract infections in humans
Enteroinvasive E. coli Actively invade colonic Large bowel diarrhea Plasmid-encoded
(EIEC) epithelial cells ­invasion genes such as
ipaH
Adherent-invasive E. coli Invade colonic epithelial cells Granulomatous/histiocytic None known
(AIEC) using unknown virulence ulcerative colitis in dogs
mechanisms (especially boxers)
Enteroaggregative E. coli Adhere to intestinal epithelium Persistent watery diarrhea in aggR gene
(EAEC) via fimbriae and aggregate in humans
bricklike fashion

A B
FIGURE 46-1  Scanning and transmission electron micrographs showing actin pedestals induced by enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC).
A, EPEC generates attaching and effacing (AE) lesions on the intestinal epithelium after infection of gnotobiotic piglets. Note the pedestal-like structures on host cells beneath attached
bacteria. (From Campellone KG, Leong JM. Tails of two Tirs: actin pedestal formation by enteropathogenic E. coli and enterohemorrhagic E. coli. Curr Opin Microbiol 2003;6(1):82-90.)
B, Actin pedestals that resemble AE lesions formed in vivo are also generated on cultured epithelial (HeLa) cells. (Courtesy of Knutton S. In: Knutton S, Rosenshine L, Pallen, MJ, et al. A novel
EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells. EMBO J. 1998;17:2166-2176.)

and water that lead to diarrhea, hypovolemia, and metabolic
acidosis. LT may also downregulate innate host immune
responses and promote ETEC adherence. In contrast, ST binds
to the extracellular domain of guanylyl cyclase C, which leads
to accumulation of intracellular cyclic GMP and ultimately
secretion of chloride and decreased absorption of NaCl, with
resultant osmotic diarrhea. Two different ST enterotoxins have
been identified, STa and STb. ST-producing ETEC have been
detected in young dogs with diarrhea.8-10
In addition to producing attaching and effacing lesions similar
to those created by EPEC, EHEC (Shiga-toxin–producing E. coli,
or STEC) produce Shiga-like toxins (also known as verotoxins).
They cause diarrhea, hemorrhagic colitis, and hemolytic-uremic
syndrome (HUS) (e.g., E. coli strain O157:H7). Shiga-like toxins
are absorbed from the intestinal lumen and cause vascular endo-
thelial damage, which may lead to a multiorgan thrombotic pro-
cess in the absence of bacteremia. EHEC can also produce other
toxins that contribute to the pathogenesis of the disease. HUS
in humans is characterized by renal failure, widespread micro-
thrombotic damage to a variety of organs, hemolytic anemia,
and sometimes diarrhea. Similar syndromes have been described
in dogs, but an associated E. coli infection was not demon-
strated.11,12 HUS has been reproduced experimentally in dogs
with a non-O157:H7 E. coli serotype.13 Based on the similarity

of renal lesions to those described in HUS, and the practice of
feeding predominantly raw meat diets to greyhounds, it has been
hypothesized that cutaneous and renal glomerular vasculopathy
of greyhounds (also known as “Alabama rot” or “Greenetrack
disease”) may result from infection with EHEC strains such as
O157:H7.14 A similar condition was described in a great Dane
from Germany.15 Some dogs develop cutaneous lesions in the
absence of renal failure; other dogs develop renal failure before
the onset of cutaneous lesions.
NTEC produce two toxins: CNF and cytolethal distending
toxin. Multiple CNF types have been identified (CNF1, CNF2,
and CNF3). NTEC have been associated with diarrhea, bacte-
remia, and urinary tract infections in human patients, as well as
with diarrhea in dogs.16
EIEC actively invade colonic epithelial cells. This, together
with the associated inflammatory response, leads to hemor-
rhagic, large bowel diarrhea. In human patients, the EIEC path-
ovar is uncommonly detected compared with EPEC and ETEC.
AIEC, which lack the virulence genes that identify EIEC, have
been associated with granulomatous colitis in boxer dogs and
infrequently in the French bulldog and Border collie. This infec-
tion typically affects young adult boxer dogs that show signs
of severe colitis, with colonic thickening and ulceration, and
weight loss that is often marked.4,17 It has been suggested that a
heritable anomaly in boxer dogs predisposes them to the infec-
tion, given the marked breed predisposition for the disease.18
The EAEC pathovar is considered an emerging pathogen
in human patients. It adheres to epithelial cells of the termi-
nal ileum and colon using fimbriae in a characteristic “stacked
brick” pattern. Expression of these fimbriae is encoded by a
plasmid gene known as aggR. Bacterial aggregation is followed
by damage and a subsequent inflammatory response, which
leads to development of persistent watery diarrhea. Occasion-
ally, EAEC strains also produce toxins, such as verocytotoxin.19
Their role in dogs and cats requires further investigation.
Physical Examination Findings
Physical examination may reveal dehydration and abdominal pain
in puppies with E. coli diarrhea. Dogs with HUS have had cutane-
ous erythema and well-demarcated, multifocal cutaneous ulcers of
the limbs. Fever and peripheral edema have also been described.
Boxer dogs with granulomatous colitis may have poor body con-
dition. A thickened and irregular rectal wall may be detected on
rectal palpation, and the feces may contain fresh blood and mucus.
Diagnosis
Diagnosis of intestinal E. coli infections, with the exception of
granulomatous colitis of boxer dogs, is difficult or impossible
with currently available diagnostic assays, because of the fact
that healthy dogs and cats shed E. coli, and it is a combination
of bacterial virulence factors and host immune competence that
contributes to the development of clinical signs. This section
therefore focuses primarily on the more specific clinical syn-
dromes caused by EHEC and AIEC.
Laboratory Abnormalities
Complete Blood Count
CBC changes in dogs with E. coli diarrhea are generally mild
and nonspecific. Leukocytosis may be present. Dogs infected
with EHEC may be thrombocytopenic and have evidence of
CHAPTER 46  Enteric Escherichia coli Infections 447
FIGURE 46-2  Ulcerated and erythematous colon of a 2-year-old intact male boxer
dog with granulomatous colitis as viewed during colonoscopy. The dog had a 7-month
history of marked weight loss and severe diarrhea, with increased frequency of defecation
and hematochezia.
microangiopathic hemolysis. Microcytic anemia may be present
in boxer dogs with granulomatous colitis.
Serum Chemistry Panel and Urinalysis
Laboratory abnormalities in dogs with E. coli diarrhea could
include electrolyte abnormalities and prerenal azotemia in dogs
with severe diarrhea. Dogs infected with EHEC have had vari-
able evidence of hypoalbuminemia, prerenal azotemia, increased
activities of ALT and creatine kinase, hematuria, proteinuria,
and isosthenuria. Boxer dogs with granulomatous colitis are
commonly hypoalbuminemic.
Diagnostic Imaging
Findings on abdominal radiography and abdominal ultrasound
are typically unremarkable or show fluid-filled intestinal loops.
Abdominal ultrasound in boxer dogs with granulomatous coli-
tis often reveals mild or moderate mesenteric or sublumbar
lymphadenomegaly, and the colonic wall can appear thickened.
Endoscopic Findings
Proctoscopy or colonoscopy is usually performed in order to
obtain colonic biopsies from boxer dogs suspected to have gran-
ulomatous colitis. Proctoscopy requires less fastidious colonic
preparation compared to colonoscopy, can be performed under
heavy sedation, and is more cost effective compared to colo-
noscopy. Most boxer dogs with granulomatous colitis have
involvement of the descending colon, underscoring the diagnos-
tic utility of proctoscopy. Common changes in appearance to
the colonic wall include erythema, irregularity, and ulceration
of the rectal wall (Figure 46-2).
Microbiologic Testing
Bacterial Isolation and Typing
E. coli is commonly isolated on routine bacteriologic media
from feces of both healthy and diarrheic dogs. A differential
medium such as MacConkey agar is often used. Apart from
dogs with granulomatous colitis, attempts to specifically iden-
tify E. coli as a cause of diarrhea are generally performed only
448 SECTION 2  Bacterial Diseases

by reference or public health laboratories when an outbreak has

occurred, or for research purposes. E. coli can be isolated from
colonic biopsies of dogs with granulomatous colitis, which
permits subsequent antimicrobial susceptibility testing.20 Sero-
logic identification, which is performed using specific O and
H antisera, can be costly and is generally performed by large
public health laboratories in epidemiologic investigations.

Virulence Assays
Immunoassays are available for detection of Shiga toxin, ST,
and LT. A Vero cell cytotoxicity assay has been used to detect
verocytotoxin. Identification of EPEC and AIEC often relies on
assays that assess adherence in tissue culture. In research set-
tings, molecular diagnostic techniques using DNA probes or
PCR assays have revolutionized the ability to detect and dif-
ferentiate between pathogenic and nonpathogenic strains of E.
coli. EPEC characteristically possess the eaeA gene. Detection of
genes that encode LT, ST, and Shiga-like toxin allows identifica- A
tion of ETEC and EHEC. EIEC are identified based on the pres-
ence of invasion-associated genes such as ipaH. The aat gene is
a diagnostic marker for EAEC detection.19

Pathologic Findings
The most significant pathologic findings in intestinal E. coli
infections occur with EHEC and AIEC infections.

EHEC
Cutaneous ulcerative lesions in dogs with suspected HUS are
characterized histopathologically by fibrinoid vascular necro-
sis, with dermal thrombosis and leukocytoclastic vasculitis.
Grossly, the kidneys may be swollen and have cortical pete-
chiae. Histopathology reveals hyaline fibrinous thrombi in
glomerular capillaries and afferent arterioles, glomerular and
multifocal tubular necrosis, and leukocytoclastic vasculitis with
fibrinoid necrosis. B

AIEC FIGURE 46-3  A, Histopathology showing severe, chronic, histiocytic colitis in a

Histopathologic lesions in boxers and French bulldogs with
granulomatous colitis are pathognomonic and include neutro-
philic inflammation, epithelial ulceration, crypt hyperplasia and
distortion, decreased goblet cell numbers, and abundant macro-
phages that stain positive with periodic acid–Schiff (PAS) stain
(Figure 46-3). The presence of E. coli within macrophages can
be confirmed using fluorescence in situ hybridization, which has
been recommended as part of the diagnostic work-up of dogs
suspected to have histiocytic ulcerative colitis.5,18
Treatment and Prognosis
Dogs and cats with severe diarrhea due to pathogenic E. coli
may require intravenous fluid therapy. Dogs with granuloma-
tous colitis can show dramatic responses to treatment with
enrofloxacin (10 mg/kg PO q24h), and a minimum treatment
duration of 8 weeks is recommended.18 Administration of fluo-
roquinolones is usually associated with rapid resolution of clini-
cal signs and resolves cellular infiltration characteristic of this
disorder on colonic biopsy. Because enrofloxacin resistance has
been documented in some isolates from dogs with granuloma-
tous colitis, attempts to isolate E. coli from colonic biopsies
before treatment is recommended so that antimicrobial suscepti-
bility testing can be performed.20 Antimicrobials that penetrate
intracellularly, such as fluoroquinolones, chloramphenicol,
2-year-old male boxer dog. The inflammatory infiltrate is comprised of large, epithelioid
macrophages and lesser numbers of neutrophils, plasma cells, and lymphocytes. B, Histio-
cytes in the lamina propria contain PAS-positive material.
rifampin, or trimethoprim-sulfonamides, should be chosen for
treatment based on the results of susceptibility testing.
Immunity and Vaccination
Currently, no vaccines for E. coli infections are available for
dogs and cats. Vaccines for EPEC, ETEC, and EHEC have been
investigated for animals and humans on a research basis,21-23
including a transdermal LT vaccine for prevention of travelers’
diarrhea.24
Prevention
The reader is referred to Chapter 11 and Chapter 45 for infor-
mation on prevention of enteric bacterial infections.
Public Health Aspects
Pathogenic E. coli that cause diarrhea have been particularly
prevalent in children in developing countries and in travel-
ers to developing countries. EHEC can be acquired following
 CHAPTER 46  Enteric Escherichia coli Infections 449

ingestion of undercooked ground hamburger meat, vegetables,
and nonchlorinated drinking and swimming water that have
been contaminated with fecal material. Infection has also been
reported following contact with animals housed in petting
zoos.25 HUS is reported as the most common cause of acute
renal failure in children. Clinical signs in humans consist of
gastrointestinal signs, often with profuse, bloody diarrhea,
abdominal cramps, pallor, weakness, and oliguria or anuria.26
Dogs have been suggested as a source of human infection with
EPEC and EHEC.27,28
The possibility of zoonotic transmission of multidrug resis-
tant E. coli that are shed by dogs and cats is an emerging con-
cern. A variety of resistance mechanisms have been identified in
E. coli isolates from dogs and cats, which include production
of efflux pumps and extended-spectrum and AmpC β-lactamase
enzymes. There is some evidence that E. coli strains shed by
healthy companion animals can be shared with humans that
reside in the same household,29-32 but more studies are required
to determine the extent to which this occurs and its zoonotic sig-
nificance. Transfer of plasmids that carry resistance genes from
canine or feline E. coli isolates and those of humans also has the
potential to occur.
For hospitalized animals, staff should use contact precau-
tions for any dog or cat suspected to have enteropathogenic
illness, including the use of warning signs, gloves, and gowns,
hand washing, and cleaning with bleach-based or accelerated
hydrogen peroxide-based disinfectants. Endoscopes should be
disinfected thoroughly between uses. Veterinarians should dis-
cuss the zoonotic implications of enteropathogenic bacterial
infection with pet owners, especially in relation to the presence
of young children or otherwise immunocompromised individu-
als in the household.

CASE EXAMPLE
Signalment: “Caesar”, 2-year-old male boxer dog from
Tulare, CA (Figure 46-4)
History: Caesar was evaluated for chronic diarrhea, which
had been present since he was 6 months of age. His feces
were loose and contained a small amount of frank blood.
He strained to defecate many times a day, and the owner
sometimes noticed mucus in the diarrhea. Every few
months, Caesar’s appetite and energy level declined and
the diarrhea became more severe. His signs transiently
improved after treatment with subcutaneous fluids and
metronidazole. The owners had tried a variety of different
diets such as fiber-supplemented diets and elimination
diets that contain novel, single protein sources, but these
strategies did not help. His current diet was a digestible
commercial diet, and his appetite was good. He was up
to date on vaccinations, including those for rabies, canine
distemper, canine parvovirus, canine adenovirus, and
Bordetella bronchiseptica. He was primarily an indoor dog
with access to a suburban backyard.
Physical Examination:
Body Weight: 23.4 kg
General: Bright, alert, and responsive. Ambulatory on all four
limbs. T = 102.5°F (39.2°C), HR = 102 beats/min, panting, FIGURE 46-4  “Caesar,” a 2-year-old male boxer dog from Tulare, CA, that was
mucous membranes pink, CRT = 1 s. diagnosed with granulomatous colitis following colonoscopy and biopsy.
Integument, Eyes, Ears, Nose, and Throat: No significant
abnormalities were noted. A moderate amount of dental MCHC 32.9 g/dL (33-36 g/dL)
calculus was present. WBC 12,590 cells/µL (6000-13,000 cells/µL)
Musculoskeletal: Body condition score was 3/9 with Neutrophils 9631 cells/µL (3000-10,500 cells/µL)
symmetrical muscling. Lymphocytes 1674 cells/µL (1000-4000 cells/µL)
All Other Systems: On rectal examination, the walls of Monocytes 781 cells/µL (150-1200 cells/µL)
the rectum were smooth. There was fresh blood on the Platelets were clumped but appeared adequate in number.
glove following palpation. No other clinically significant Serum Chemistry Profile:
abnormalities were noted. Sodium 155 mmol/L (145-154 mmol/L)
Laboratory Findings: Potassium 4.2 mmol/L (3.6-5.3 mmol/L)
CBC: Chloride 116 mmol/L (108-118 mmol/L)
HCT 54.4% (40%-55%) Bicarbonate 23 mmol/L (16-26 mmol/L)
MCV 76.8 fL (65-75 fL) Phosphorus 4.4 mg/dL (3.0-6.2 mg/dL)
450 SECTION 2  Bacterial Diseases
Calcium 10.4 mg/dL (9.7-11.5 mg/dl)
BUN 18 mg/dL ( 5-21 mg/dL)
Creatinine 1.1 mg/dL (0.3-1.2 mg/dL)
Glucose 105 mg/dL (64-123 mg/dL)
Total protein 6.5 g/dL (5.4-7.6 g/dL)
Albumin 2.9 g/dL (3.0-4.4 g/dL)
Globulin 3.6 g/dL (1.8-3.9 g/dL)
ALT 68 U/L (19-67 U/L)
AST 46 U/L (19-42 U/L)
ALP 86 U/L (21-170 U/L)
GGT 5 U/L (0-6 U/L)
Cholesterol 200 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL).
Fecal Examination: Centrifugal fecal flotation: Negative for
parasites.
Colonoscopy: The entire mucosal surface of the colon was
erythematous and irregular, with multifocal areas of mucosal
ulceration (see Figure 45-1).
SUGGESTED READINGS
Sherman PM, Ossa JC, Wine E. Bacterial infections: new and emerging
enteric pathogens. Curr Opin Gastroenterol. 2010;26(1):1-4.
Simpson KW, Dogan B, Rishniw M, et al. Adherent and invasive Esch-
erichia coli is associated with granulomatous colitis in boxer dogs.
Infect Immun. 2006;74(8):4778-4792.
REFERENCES
1. Escherich T. Die Darmbakterien des Neugeborenen und Säuglings.
Fortschr Med. 1885;3:515-522, 547-554.
2. Sancak AA, Rutgers HC, Hart CA, et al. Prevalence of enteropathic
Escherichia coli in dogs with acute and chronic diarrhea. Vet Rec.
2004;154(4):101-106.
3. Morato EP, Leomil L, Beutin L, et  al. Domestic cats constitute
a natural reservoir of human enteropathogenic Escherichia coli
types. Zoonoses Public Health. 2009;56(5):229-237.
4. Simpson KW, Dogan B, Rishniw M, et al. Adherent and invasive
Escherichia coli is associated with granulomatous colitis in boxer
dogs. Infect Immun. 2006;74(8):4778-4792.
5. Celli J, Deng W, Finlay BB. Enteropathogenic Escherichia coli
(EPEC) attachment to epithelial cells: exploiting the host cell cyto-
skeleton from the outside. Cell Microbiol. 2000;2(1):1-9.
6. Morato EP, Leomil L, Beutin L, et  al. Domestic cats constitute
a natural reservoir of human enteropathogenic Escherichia coli
types. Zoonoses Public Health. 2009;56:229-237.
7. de Almeida PM, Arais LR, Andrade JR, et al. Characterization of
atypical Enteropathogenic Escherichia coli (aEPEC) isolated from
dogs. Vet Microbiol. 2012;158:420-424.
8. Drolet R, Fairbrother JM, Harel J, et al. Attaching and effacing and
enterotoxigenic Escherichia coli associated with enteric colibacil-
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9. Hammermueller J, Kruth S, Prescott J, et  al. Detection of toxin
genes in Escherichia coli isolated from normal dogs and dogs with
diarrhea. Can J Vet Res. 1995;59(4):265-270.
10. Beutin L. Escherichia coli as a pathogen in dogs and cats. Vet Res.
1999;30(2-3):285-298.
11. Chantrey J, Chapman PS, Patterson-Kan JC. Haemolytic-­uraemic
syndrome in a dog. J Vet Med A Physiol Pathol Clin Med.
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12. Dell’Orco M, Bertazzolo W, Pagliaro L, et  al. Hemolytic-uremic
syndrome in a dog. Vet Clin Pathol. 2005;34(3):264-269.
Histopathology of Colonic Biopsies: Severe, chronic,
histiocytic and ulcerative colitis. Histiocytes in the lamina
propria contained PAS-positive material (see Figure 46-3).
Diagnosis: Granulomatous colitis of boxer dogs
Treatment: Enrofloxacin, 6 mg/kg PO q24h for 8 weeks and
a commercial low-residue dry and canned dog food. This
was associated with clinical improvement within a week
after starting the medication. At a recheck 4 weeks later, the
owner reported that Caesar’s feces were completely normal
in consistency and frequency.
Comments: In this dog, the diagnosis of granulomatous colitis
was made solely based on histopathologic findings. Culture
of a biopsy specimen could also have been performed and is
now recommended given the emergence of fluoroquinolone-
resistant AIEC strains of E. coli.
13. Wang JY, Wang SS, Yin PZ. Haemolytic-uraemic syndrome caused
by a non-O157:H7 Escherichia coli strain in experimentally inocu-
lated dogs. J Med Microbiol. 2006;55(Pt 1):23-29.
14. Cowan LA, Hertzke DM, Fenwick BW, et al. Clinical and clinico-
pathologic abnormalities in greyhounds with cutaneous and renal
glomerular vasculopathy: 18 cases (1992-1994). J Am Vet Med
Assoc 210(6):789–793.
15. Rotermund A, Peters M, Hewicker-Trautwein M, et  al. Cutane-
ous and renal glomerular vasculopathy in a great Dane resembling
“Alabama rot” of greyhounds. Vet Rec. 2002;151(17):510-512.
16. Starcic M, Johnson JR, Stell AL, et  al. Haemolytic Escherichia
coli isolated from dogs with diarrhea have characteristics of
both uropathogenic and necrotoxigenic strains. Vet Microbiol.
2002;85(4):361-377.
17. Van Kruiningen HJ, Civco IC, Cartun RW. The comparative
importance of E. coli antigen in granulomatous colitis of Boxer
dogs. APMIS. 2005;113(6):420-425.
18. Mansfield CS, James FE, Craven M, et al. Remission of histiocytic
ulcerative colitis in Boxer dogs correlates with eradication of invasive
intramucosal Escherichia coli. J Vet Intern Med. 2009;23(5):964-969.
19. Scavia G, Staffolani M, Fisichella S, et al. Enteroaggregative Esch-
erichia coli associated with a foodborne outbreak of gastroenteritis.
J Med Microbiol. 2008;57(Pt 9):1141-1146.
20. Craven M, Dogan B, Schukken A, et  al. Antimicrobial resistance
impacts clinical outcome of granulomatous colitis in boxer dogs.
J Vet Intern Med. 2010;24(4):819-824.
21. Gu J, Liu Y, Yu S, et  al. Enterohemorrhagic Escherichia coli tri-
valent recombinant vaccine containing EspA, intimin and Stx2
induces strong humoral immune response and confers protection in
mice. Microbes Infect. 2009;11(10-11):835-841.
22. Keller R, Hilton TD, Rios H, et  al. Development of a live oral
attaching and effacing Escherichia coli vaccine candidate using
Vibrio cholerae CVD 103-HgR as antigen vector. Microb Pathog.
2010;48(1):1-8.
23. Rojas RL, Gomes PA, Bentancor LV, et  al. Salmonella enterica
serovar Typhimurium vaccine strains expressing a nontoxic Shiga-
like toxin 2 derivative induce partial protective immunity to the
toxin expressed by enterohemorrhagic Escherichia coli. Clin Vac-
cine Immunol. 2010;17(4):529-536.
24. Frech SA, Dupont HL, Bourgeois AL, et al. Use of a patch contain-
ing heat-labile toxin from Escherichia coli against traveller’s diar-
rhoea: a phase II, randomized, double-blind, placebo-controlled
field trial. Lancet. 2008;371(9629):2019-2025.
 CHAPTER 46  Enteric Escherichia coli Infections
25. Sherman PM, Ossa JC, Wine E. Bacterial infections: new and emerg-
ing enteric pathogens. Curr Opin Gastroenterol. 2010;26(1):1-4.
26. Scheiring J, Andreoli SP, Zimmerhackl LB. Treatment and outcome
of Shiga-toxin-associated hemolytic-uremic syndrome. Pediatr
Nephrol. 2008;23(10):1749-1760.
27. Nakazato G, Gyles C, Ziebell K, et al. Attaching and effacing Esch-
erichia coli isolated from dogs in Brazil: characteristics and sero-
typic relationship to human enteropathogenic E. coli (EPEC). Vet
Microbiol. 2004;101(4):269-277.
28. Hogg RA, Holmes JP, Ghebrehewet S, et  al. Probable zoonotic
transmission of verocytotoxigenic Escherichia coli O157 by dogs.
Vet Rec. 2009;164(10):304-305.
29. Harada K, Okada E, Shimuzu T, et  al. Antimicrobial resistance,
virulence profiles and phylogenetic groups of fecal Escherichia coli
isolates: a comparative analysis between dogs and their owners in
Japan. Comp Immunol Microbiol Infect Dis. 2012;35:139-144.
451
30. Stenske KA, Bemis DA, Gillespie BE, et al. Comparison of clonal
relatedness and antimicrobial susceptibility of Escherichia coli from
healthy dogs and their owners. Am J Vet Res. 2009;70:1108-1116.
31. Johnson JR, Clabots C, Kuskowski MA. Multiple-host sharing,
long-term persistence, and virulence of Escherichia coli clones
from human and animal household members. J Clin Microbiol.
2008;46:4078-4082.
32. Damborg P, Nielsen SS, Guardabassi L. Escherichia coli shedding
patterns in humans and dogs: insights into within-household trans-
mission of phylotypes associated with urinary tract infections. Epi-
demiol Infect. 2012;137:1457-1464.
CHAPTER 47
Campylobacteriosis
Jane E. Sykes and Stanley L. Marks
Overview of Campylobacteriosis
First Described: Germany, 1886 (Theodor Escherich)1; the
organism was not isolated until the 1950s
Cause: Campylobacter spp. (curved to spiral gram-negative
bacteria that belong to the Campylobactereaceae)
Affected Hosts: Humans and a large variety of other animal
species
Geographic Distribution: Worldwide
Primary Mode of Transmission: Fecal-oral
Major Clinical Signs: Fever, lethargy, anorexia, large bowel
diarrhea
Differential Diagnoses: Differential diagnoses for suspected
Campylobacter enterocolitis include canine and feline par-
vovirus infection, canine distemper virus infection, salmon
­poisoning disease, Escherichia coli infection, clostridial
diarrhea, salmonellosis, giardiasis, tritrichomoniasis (cats),
cryptosporidiosis, whipworms, leptospirosis, dietary indis-
cretion, gastrointestinal foreign body, pancreatitis, inflam-
matory bowel disease, lymphoma, ­hyperthyroidism (cats),
hypoadrenocorticism, toxins (including drugs).
Human Health Significance: Important zoonosis. Dogs and cats
may be a source of human infection with Campylobacter,
some of which may be resistant to antimicrobial drugs.
Etiology and Epidemiology
Campylobacter are thin, gram-negative, curved, S-shaped or
spiral rods that are often motile by means of a single polar
flagellum at one or both ends (Figure 47-1). They are micro-
aerophilic (i.e., they grow best when levels of oxygen are lower
than that present in the atmosphere), and are widespread in the
gastrointestinal tracts of animals. Campylobacter spp. have the
potential to be zoonotic and are an important cause of food-
borne illness in humans, especially in association with poultry
products. They can survive weeks to months in water. More
than a dozen species have been identified in dogs and cats (Box
47-1). All species are commonly isolated from both healthy and
diarrheic dogs and cats. The prevalence of shedding by clinically
healthy dogs, for example, ranges from 15% to 87%,2 and as
high as 58% of non-diarrheic and diarrheic cats shed Campy-
lobacter as determined by PCR assay.3 Campylobacter jejuni is
most commonly incriminated as a cause of diarrhea. The evi-
dence for disease causation by other species, such as Campylo-
bacter upsaliensis and Campylobacter helveticus, is less certain,
because the isolation rates of these species are often similar in
452
healthy and diarrheic dogs and cats. In one study, Campylo-
bacter species were isolated more commonly from healthy cats
than from cats with diarrhea.3
C. jejuni, C. helveticus, and C. upsaliensis are the species
most frequently isolated from the stool of dogs and cats, with
C. helveticus and C. upsaliensis being the predominant isolates
in some studies.3,4 Other commonly identified species in dogs
and cats include Campylobacter coli, C. helveticus, and Campy-
lobacter lari. In one study, up to 12 Campylobacter species were
present simultaneously in dogs,5 and coinfection with multiple
Campylobacter species was detected in 47% of 74 cats from
which Campylobacter was isolated.3 The prevalence of Campy-
lobacter carriage is particularly high in young dogs and cats and
those housed with large numbers of other animals such as in
kennels or shelters. Increased isolation rates are also observed in
the spring, summer, and fall months, depending on study loca-
tion.6,7 Cats less than 1 year of age were significantly more likely
to shed Campylobacter than cats older than a year of age, and
median duration of shedding was around 6 weeks in a study from
the upper Midwestern United States.7 Young age and the feeding
of homemade cooked food to dogs was associated with Campy-
lobacter carriage in dogs from eastern Canada.2 Ownership of
puppies and kittens is a significant risk factor for development
of campylobacteriosis by humans, especially young children.8,9
Clinical Features
Signs and Their Pathogenesis
Campylobacter colonize the lower intestinal tract, including the
jejunum, ileum, and colon. In contrast to other enteropathogenic
bacteria, Campylobacter possess relatively few virulence factors,
and host factors are important in determining the severity of
clinical signs that develop. Campylobacter can invade intestinal
epithelial cells and produce cytolethal distending toxin, which
causes cell cycle arrest and apoptosis, and in humans, stimulates
Il-8 production, which leads to an inflammatory response. The
organism can produce a polysaccharide capsule, which may also
be a virulence factor.10 Puppies and kittens less than 6 months
of age are most likely to show diarrhea in association with Cam-
pylobacter infection, and stress, overcrowding, and concurrent
gastrointestinal infections with other bacteria, protozoa, and hel-
minth parasites may also contribute to clinical signs. The sever-
ity of diarrhea can range from mild diarrhea with loose stools
through to watery, bloody, or mucoid diarrhea, with lethargy,
inappetence, and, less commonly, vomiting. Other signs of large
bowel diarrhea such as tenesmus may also be present. Fever may
occur in severe acute campylobacteriosis in puppies and kittens.
Diarrhea is generally self-limiting within a 1- to 2-week period,
although chronic diarrhea can also occur in dogs.11,12 Rarely,
extraintestinal Campylobacter infections in dogs have been
 CHAPTER 47  Campylobacteriosis 453

ATL
0
1
GB 2
FF 3
4

5
6

FIGURE 47-1  Scanning electron micrograph of Campylobacter. (From Centers for Dis-
FIGURE 47-2  Abdominal ultrasound image from a 10-year-old intact male ­Brittany
ease Control and Prevention, Atlanta, GA.)
spaniel with Campylobacter bacteremia, gastroenteritis, and probable cholecystitis, ­showing
thickening and irregularity of the gallbladder (GB) wall and free abdominal fluid (FF).

BOX 47-1 Diagnostic Imaging

Findings on plain abdominal radiography and abdominal ultra-
Campylobacter Species Described in Dogs and Cats sound are typically unremarkable or show fluid-filled intestinal
loops. A thickened gallbladder wall may be seen using ultrasound
C. jejuni in animals with Campylobacter cholecystitis (Figure 47-2).
C. upsaliensis
C. lari Microbiologic Testing
C. coli Microbiologic assays available for diagnosis of campylobacteri-
C. helveticus osis in dogs and cats include microscopic examination, culture,
C. concisus and PCR assays (Table 47-1). Because of the high prevalence of
C. fetus Campylobacter colonization in healthy dogs and cats, it is dif-
C. gracilis ficult to interpret the significance of positive test results on feces.
C. mucosalis The results must always be considered in light of the patient
C. showae signalment, history, clinical signs, and exclusion of other causes
C. sputorum of diarrhea. Specific identification of C. jejuni, as opposed to
C. curvus other Campylobacter species, may also add support to the role
C. hyointestinalis of Campylobacter in disease.
C. rectus
Microscopic Examination
Although not diagnostic for campylobacteriosis, a direct fecal
smear may reveal large numbers of fine, S-shaped or gull-shaped
reported, specifically cholecystitis, bacteremia, and abortion.13,14 organisms following staining with Gram or Romanowsky stains
These have also been described in humans.15,16 (Figure 47-3). Detection of these organisms only suggests the
presence of Campylobacter-like organisms and should not be
Physical Examination Findings used as the sole method to diagnose campylobacteriosis because
Physical examination findings in severely affected young ani- of the inability to differentiate between similar-appearing
mals with campylobacteriosis are variable and include lethargy, organisms such as Arcobacter or nonpathogenic campylobac-
dehydration, fever, and abdominal pain. Diarrhea, sometimes ters. Fecal leukocytes may also be present. More advanced
with fresh blood or mucus, may be found on rectal examination microscopic techniques that have been used to identify Cam-
in dogs. pylobacter in the feces include darkfield and phase-contrast
microscopy, which are used on fresh fecal specimens and show
Diagnosis the characteristic morphology and darting motility of the organ-
ism. These are generally not performed routinely in clinical situ-
Laboratory Abnormalities ations and require significant technical expertise.
Complete Blood Count and Chemistry Panel
Basic laboratory testing in puppies and kittens with Campylo- Bacterial Isolation
bacter diarrhea generally reveals mild and nonspecific changes. Campylobacter cannot be isolated on routine bacteriological
A leukocytosis may be present. Animals with systemic cam- media. Fecal enteric panels designed to detect bacterial entero-
pylobacteriosis and cholecystitis may show neutrophilia or pathogens in feces generally include isolation on selective
neutropenia with increased circulating band neutrophils and media such as charcoal- or blood-based Campylobacter media,
biochemical evidence of cholestasis and hepatic dysfunction (see which often contain antimicrobials to eliminate other bacteria
Case Example). (such as cefoperazone). The inoculated medium is incubated
454 SECTION 2  Bacterial Diseases
TABLE 47-1
Diagnostic Assays Available for Campylobacteriosis in Dogs and Cats
Assay Specimen Type Target
Bacterial isolation Feces, blood, bile, Campylobacter
intestinal tissues organism
collected at
n­ecropsy
Polymerase chain As for isolation Campylobacter
reaction DNA
FIGURE 47-3  Cytology showing Campylobacter organisms. Gram stain, 1000×
magnification.
in a microaerophilic environment for 72 to 96 hours. Incuba-
tion is frequently carried out at 42°C to select for thermophilic
­Campylobacter; however, a temperature of 37°C should also be
used to ensure isolation of variable or nonthermophilic species.
The use of direct plating onto charcoal-based media may be most
sensitive for isolation of Campylobacter from pets, but use of a
combination of isolation methods increases sensitivity further.17
Some strains require more hydrogen than others and do not
grow under routine conditions used for Campylobacter isola-
tion. Species identification can then be performed on the basis
of biochemical testing or the use of PCR followed by sequencing
of the PCR product. Biochemical testing can be highly variable,
resulting in inaccurate identification. Serotyping has been tradi-
tionally used to identify strains of C. jejuni for epidemiologic
studies, and more recently, molecular methods such as pulsed-
field gel electrophoresis and PCR-based typing methods (includ-
ing multilocus sequence typing), have been used.18-20 Techniques
based on matrix-assisted laser desorption/ionization-time-of-
flight (MALDI-TOF) mass spectrometry also show promise for
Performance
False negatives may occur, especially following antimicrobial therapy,
and some species may not grow under routine conditions used to
isolate Campylobacter. Isolation from feces does not i­mply that
Campylobacter is the cause of disease. Allows subsequent antimi-
crobial susceptibility testing.
Rapid (within hours); can be highly sensitive and specific. Positive
results from feces do not imply that Campylobacter is the cause of
disease. Some assays do not differentiate between Campylobacter
species, but others are species specific (e.g., only Campylobacter
jejuni or only Campylobacter coli). Check with the laboratory to
determine what assays are offered.
rapid identification of Campylobacter species and strains (see
Chapter 3).21,22 Isolation of Campylobacter in culture permits
subsequent antimicrobial susceptibility testing, although this is
not routinely performed by most veterinary laboratories.
Molecular Diagnosis Using the Polymerase Chain Reaction
Both conventional and real-time PCR-based assays have been
developed for direct detection of Campylobacter in stool, and
some are available commercially for use in dogs and cats. An
advantage of PCR is that it can detect species that grow poorly
in culture, and it overcomes problems relating to overgrowth
of other fecal bacteria.3 Both Campylobacter genus-specific and
species-specific assays (such as those that only detect C. jejuni)
have been developed. In human patients, a real-time PCR assay
for detection of C. jejuni was at least as sensitive as culture.23
Pathologic Findings
Gross Pathologic Findings
Gross necropsy findings in puppies with campylobacteriosis
include fluid-filled intestinal loops with mucosal congestion and
edema, especially within the distal jejunum, ileum, cecum, and
colon.12,24 Mesenteric lymphadenomegaly may also be present.
Histopathologic findings
Campylobacter infection can result in villous blunting and
fusion, epithelial hyperplasia, and congestion and lymphoplas-
macytic inflammation of the lamina propria. Crypts contain
filamentous bacteria that stain with silver stains such as War-
thin-Starry (Figure 47-4). Immunohistochemistry can be used to
identify Campylobacter within intestinal crypts.12
Treatment and Prognosis
Uncomplicated campylobacteriosis is generally self-limiting and
resolves with supportive therapy. Because isolation of Campylo-
bacter even from diarrheic feces does not necessarily imply causa-
tion for the clinical signs, specific treatment may not be warranted
and may further disrupt the intestinal microflora. Treatment could
be considered for severely ill dogs and cats. Macrolides (such as
erythromycin or azithromycin) and fluoroquinolones (such as
enrofloxacin, 5 mg/kg q24h for 5 to 7 days) are often efficacious,
although resistance to these antimicrobials has been documented
in some Campylobacter isolates.25,26 Treatment failures could

reflect resistance to the antimicrobial used or infection with a
nonpathogenic Campylobacter species and persistence of clinical
signs from another unidentified cause. Recrudescence of infection
has been documented in human patients after treatment.27
Immunity and Vaccination
Innate, humoral, and cell-mediated immune responses are
critical for clearance of Campylobacter infection.28 Short-term
protection that is somewhat serotype specific can result from
frequent exposure to Campylobacter, but immunity wanes with
time, and reinfection is possible. Considerable effort has been
expended into development of vaccines for humans and poul-
try.29-31 No vaccine currently exists for prevention of Campylo-
bacter infection in dogs and cats.
Public Health Aspects
Campylobacter is the most common bacterial cause of enteric
illness in humans worldwide. Over 90% of human campylo-
bacteriosis in industrialized countries results from consumption
of contaminated chicken products, as well as beef and milk.
A B
FIGURE 47-4  Histopathology of an intestinal section from a 3-month-old Chihuahua puppy with Campylobacter jejuni–associated diarrhea. Abundant filamentous, spiral bacteria
pack the crypts and there is an associated lymphoplasmacytic enterocolitis. A, H&E stain; B, Giemsa stain; C, Warthin-Starry silver stain.
CASE EXAMPLE
Signalment: “Henry,” a 10-year-old intact male Brittany
spaniel from Dixon in northern California
History: Henry was brought to an emergency clinic for a 1-day
history of anorexia, lethargy, and fever. The owner, a retired
veterinarian, noted that Henry’s rectal temperature was 106°F
(41.1°C). There had been no vomiting or diarrhea, and the dog
had been drinking normally. Henry was a field trial dog and
was hunting a week before becoming ill. He was housed in
an outdoor run with no access to foreign objects other than
gravel. There were nine other apparently healthy dogs in the
household. The dogs were fed a commercial dry and canned
diet.
Physical Examination:
Body Weight: 17.8 kg
General: Quiet, alert and responsive, 5% to 7% dehydrated.
Ambulatory. T = 106.4°F (41.3°C), HR = 140 beats/min, RR =
40 breaths/min, mucous membranes pale pink and tacky,
CRT = 2 s.
CHAPTER 47  Campylobacteriosis 455
Drinking water and swimming water may also be a source of
infection. To a lesser extent, contact with other food animal spe-
cies, wild birds, and pet dogs and cats has been associated with
human campylobacteriosis.32 Ingestion of as few as 500 organ-
isms can lead to infection. When clinical signs occur, early signs
develop after an incubation period of 1 to 7 days and include
fever, myalgia, vomiting, and headache, which generally last
1 to 3 days, followed by up to one week of watery to bloody diar-
rhea and abdominal pain. Illness may be mild to severe, requiring
hospitalization. Severe illness is more likely to occur in the immu-
nosuppressed. Reactive arthritis and Guillain-Barré syndrome,
an acute inflammatory demyelinating polyneuropathy, are late-
onset immune-mediated consequences that can sometimes occur
in association with infection and may persist for weeks or years.32
In fact, C. jejuni is the most common infection that precedes Guil-
lain-Barré syndrome, which occurs in 1 in 1000 infected individu-
als.25 Extraintestinal infections that have been described include
bacteremia, hepatitis, cholecystitis, pancreatitis, peritonitis, abor-
tion and neonatal sepsis, urinary tract infections, myocarditis,
and meningitis, although these are very rare in immunocompe-
tent individuals. Campylobacter enteritis has also been identified
as a risk factor for inflammatory bowel disease.33
C
Integument, Eyes, Ears, Nose, and Throat: A large amount of
dental calculus was noted. There was moderate waxy debris
in both ear canals and pinnal erythema.
Musculoskeletal: Body condition score was 4/9, and the dog
was ambulatory with symmetrical muscling.
Cardiovascular: The femoral pulses were fair and synchronous.
No murmurs or arrhythmias were auscultated.
Respiratory: Harsh breath sounds were bilaterally noted on
thoracic auscultation, which were loudest in the ventral lung
fields.
Gastrointestinal and Genitourinary: The abdomen was
tense on palpation and moderate hepatomegaly was noted.
The urinary bladder was large. No abnormalities were
detected on rectal examination.
Lymph Nodes: All were within normal limits.
Laboratory Findings:
CBC:
HCT 42.9% (40%-55%)
MCV 70 fL (65-75 fL)
MCHC 34.5 g/dL (33-36 g/dL)
456 SECTION 2  Bacterial Diseases

The stomach and duodenal wall were mildly thickened.

Moderate mesenteric lymphadenopathy was identified,
particularly around the liver and ileocolic junction. The
prostate was enlarged with multiple small parenchymal
cysts, but was symmetric and smoothly margined. A sample
of orange, somewhat turbid abdominal fluid and an aspirate
of a mesenteric lymph node were obtained using ultrasound
guidance.
Cytology Findings: Abdominal fluid: Modified transudate.
TP = 4.8 g/dL, 20,000 RBC/µL, 460 nucleated cells/µL, with
72% neutrophils, 21% small mononuclear cells, and 7%
large mononuclear cells. The nucleated cells were primarily
nondegenerate neutrophils with smaller numbers of
lymphocytes and macrophages. The lymphocytes were a
heterogenous population of small mature lymphocytes
and scattered granular lymphocytes. Several pyknotic cells
and rare erythrophagocytic macrophages were present. No
FIGURE 47-5  Abdominal radiograph from a 10-year-old intact male Brittany
spaniel with Campylobacter bacteremia, gastroenteritis, and probable cholecystitis infectious organisms were seen.
that shows hepatomegaly and corrugation of the colonic wall. Mesenteric lymph node: Mild lymphoid reactivity.
Microbiologic Testing: Vector-borne disease serologic
2 nucleated red blood cells/100 WBC testing (IFA): Positive for serum Babesia canis antibodies
WBC 6600 cells/µL (6,000-13,000 cells/µL) (1:160), negative for antibodies to Rickettsia rickettsii,
Neutrophils 2904 cells/µL (3000-10,500 cells/µL) Anaplasma phagocytophilum, and Ehrlichia canis.
Band neutrophils 2838 cells/µL Aerobic and anaerobic bacterial culture of abdominal fluid: No
Metamyelocytes 330 cells/µL growth.
Lymphocytes 330 cells/µL (1000-4000 cells/µL) Aerobic bacterial urine culture (cystocentesis specimen): No
Monocytes 198 cells/µL (150-1200 cells/µL) growth.
Eosinophils 0 cells/µL (0-1500 cells/µL) Aerobic and anaerobic blood culture (four specimens collected
Platelets 144,000/µL (150,000-400,000 platelets/µL). over 5 hours, the first inoculated into a blood culture bottle
Neutrophils, band neutrophils, and metamyelocytes and the remainder into isolator tubes): Direct smear of blood
showed mild toxic changes. culture bottle contents showed rare small curved gram-neg-
Serum Chemistry Profile: ative rods. Anaerobic culture of first specimen showed small
Sodium 146 mmol/L (145-154 mmol/L) curved gram-negative rods suspicious for Campylobacter
Potassium 4.1 mmol/L (3.6-5.3 mmol/L) spp. Partial sequencing of the 16S rRNA gene revealed 100%
Chloride 116 mmol/L (108-118 mmol/L) identity to Campylobacter jejuni. All other specimens yielded
Bicarbonate 15 mmol/L (16-26 mmol/L) no growth.
Phosphorus 3.9 mg/dL (3.0-6.2 mg/dL) Fecal enteric panel: Clostridium difficile TcdA and TcdB toxin ELISA
Calcium 8.6 mg/dL (9.7-11.5 mg/dl) negative. ELISA negative for Clostridium perfringens entero-
BUN 21 mg/dL ( 5-21 mg/dL) toxin. No Salmonella spp. cultured. No Clostridium difficile cul-
Creatinine 0.8 mg/dL (0.3-1.2 mg/dL) tured. Small numbers of Campylobacter spp. cultured (C. jejuni
Glucose 86 mg/dL (64-123 mg/dL) using partial 16S rRNA gene sequencing).
Total protein 5.0 g/dL (5.4-7.6 g/dL) Diagnosis: Campylobacter jejuni bacteremia and probable
Albumin 2.7 g/dL (3.0-4.4 g/dL) cholangiohepatitis/cholecystitis
Globulin 2.3 g/dL (1.8-3.9 g/dL) Treatment and Outcome: Henry was hospitalized and
ALT 794 U/L (19-67 U/L) treated aggressively with intravenous fluids, and before
AST 275 U/L (19-42 U/L) culture results became available, enrofloxacin (5 mg/kg
ALP 461 U/L (21-170 U/L) IV q12h) and ampicillin (22 mg/kg IV q8h). Shortly after
GGT 37 U/L (0-6 U/L) admission he vomited and developed mucus-containing
Cholesterol 273 mg/dL (135-361 mg/dL) diarrhea, and subsequently melena was observed. Over
Total bilirubin 0.7 mg/dL (0-0.2 mg/dL). the next 48 hours his temperature gradually normalized,
Urinalysis: SGr 1.013 (after fluid bolus), pH 7.5, 2+ protein but he continued to vomit intermittently. Neutropenia
(SSA), 1+ bilirubin, 1+ hemoprotein, no glucose, rare WBC/ persisted, and his platelet count dropped to 105,000. He
HPF, rare RBC/HPF, rare granular casts. also became icteric, and there were progressive increases
Imaging Findings: in the activities of liver enzymes (ALT 1179 U/L, AST 946
Thoracic and Abdominal Radiographs: The cardiovascular U/L, ALP 1088 U/L, GGT 15 U/L, total bilirubin 3.9 g/dL). By
and pulmonary structures were within normal limits. The day 4 after presentation, vomiting and diarrhea ceased and
liver was moderately enlarged. The colon was mildly gas he began eating a bland diet. Laboratory testing showed
distended and had a corrugated appearance (Figure 47-5). resolution of neutropenia and thrombocytopenia. He was
Abdominal Ultrasound: There was moderate enlargement of discharged from the hospital and treated with enrofloxacin
the liver and spleen. The gallbladder wall was thickened and (10 mg/kg PO q24h) for 4 weeks. One week after discharge,
mildly irregular. There was moderate peritoneal effusion. Henry was doing well and had a normal appetite and

activity level. The owner reported that another dog in the
household subsequently developed vomiting and diarrhea,
and Campylobacter spp. were cultured from the dog’s feces.
Comments: This was an unusual case of C. jejuni bacteremia
and probable cholangiohepatitis/cholecystitis in a dog that
required blood culture for definitive diagnosis. Culture of bile
SUGGESTED READINGS
Butzler JP. Campylobacter, from obscurity to celebrity. Clin Microbiol
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Chaban B, Ngeleka M, Hill JE. Detection and quantification of 14 Cam-
pylobacter species in pet dogs reveals an increase in species richness in
feces of diarrheic animals. BMC Microbiol. 2010;10:73.
Dasti JI, Tareen AM, Lugert R, et  al. Campylobacter jejuni: a brief
overview on pathogenicity-associated factors and disease-mediating
mechanisms. Int J Med Microbiol. 2010;300(4):205-211.
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14. Bulgin MS, Ward AC, Sriranganathan N, et al. Abortion in the dog
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16. Udayakumar D, Sanaullah M. Campylobacter cholecystitis. Int J
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25. Butzler JP. Campylobacter, from obscurity to celebrity. Clin Micro-
biol Infect. 2004;10(10):868-876.
26. Lindow JC, Poly F, Tribble DR, et  al. Caught in the act: in  vivo
development of macrolide resistance to Campylobacter jejuni infec-
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experimental infection with a well-characterized organism. Clin
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28. Janssen R, Krogfelt KA, Cawthraw SA, et al. Host-pathogen inter-
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29. Jagusztyn-Krynicka EK, Łaniewski P, Wyszyńska A. Update on
Campylobacter jejuni vaccine development for preventing human
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30. Tribble DR, Baqar S, Carmolli MP, et  al. Campylobacter jejuni
strain CG8421: a refined model for the study of campylobacteriosis
and evaluation of Campylobacter vaccines in human subjects. Clin
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31. Buckley AM, Wang J, Hudson DL, et  al. Evaluation of live-­
attenuated Salmonella vaccines expressing Campylobacter antigens for
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32. Dasti JI, Tareen AM, Lugert R, et al. Campylobacter jejuni: a brief
overview on pathogenicity-associated factors and disease-mediating
mechanisms. Int J Med Microbiol. 2010;300(4):205-211.
33. Kalischuk LD, Buret AG. A role for Campylobacter jejuni–induced
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Liver Physiol. 2010;298(1):G1-G9.
CHAPTER 48

Enteric Clostridial Infections

Jane E. Sykes and Stanley L. Marks

veterinary hospitals.4,5 In one study, living with an immuno-

Overview of Clostridial Diarrhea
First Described: 1978 (C. difficile, causative role in pseudo-
membranous colitis in humans)1; 1892 (C. perfringens, by
Nuttall and Welch).2 Evidence for a role of C. perfringens in
food-borne illness was gathered over 50 years later.3
Cause: Clostridium difficile and Clostridium perfringens (gram-
positive, spore-forming anaerobic rods)
Affected Hosts: Humans and a variety of other animals
Geographic Distribution: Worldwide
Primary Mode of Transmission: Fecal-oral; disease can result
from toxin production by organisms resident in the gas-
trointestinal tract
Major Clinical Signs: Lethargy, anorexia, diarrhea, occasion-
ally fever
Differential Diagnoses: Differential diagnosis for suspected
clostridial enterocolitis include canine and feline parvo-
virus infection, canine distemper virus infection, salmon
poisoning disease, campylobacteriosis, salmonellosis,
giardiasis, tritrichomoniasis (cats), cryptosporidiosis, whip-
worms, leptospirosis, dietary indiscretion, gastrointestinal
foreign body, pancreatitis, inflammatory bowel disease,
lymphoma, hypoadrenocorticism, toxins (including drugs)
Human Health Significance: Dogs and cats have the potential
to be a source of human infection with C. difficile, because
they can harbor strains (ribotypes) that have been
detected in humans. The role of dogs and cats as a source
of C. perfringens infection is poorly understood.
compromised human was a risk factor for C. difficile infection
in dogs, but strains present in dogs differed from those in the
household environment.6 Dogs that visit human health care
facilities are also at risk of acquiring C. difficile infections.7 The
emergence of “hypervirulent” C. difficile strains in people has
resulted in an increased incidence of disease, increased mortality
rates, increased relapse rates, and recognition of community-­
associated disease.8 A similar change has not been observed
in animals; however, a hypervirulent strain (North American
pulsotype 1 or NAP1) was identified in a dog.9 C. difficile
strains have been classified on the basis of PCR-based typing
of their ribosomal RNA genes (PCR-ribotyping). In one study,
multiple animals in shelter environments were colonized with
a single PCR-ribotype (SLO 066, 045, and 010 in three differ-
ent ­shelters, respectively).10 More detailed genetic analysis of
the C. difficile strains isolated by multilocus variable number
of tandem repeat analysis (MLVA) suggested that horizontal
transmission of strains 045 and 010 occurred within the shelter.
C. perfringens is especially widespread in soil and dust and
consists of five biotypes, A to E, based on the possession of
one or more of four major toxin genes (encoding alpha, beta,
­epsilon, and iota toxins). Each biotype can also express a subset
of at least 10 other established toxins, including C. perfringens
enterotoxin (CPE). Almost all infections in dogs and cats are
C. perfringens biotype A, which also causes the majority of clos-
tridial food-borne illness in humans. The zoonotic potential of
C. difficile and C. perfringens is still incompletely understood.

Etiology and Epidemiology

Clostridium difficile and Clostridium perfringens are gram-­
positive, spore-forming, obligately anaerobic rods that are
capable of toxin production. Clostridial spores are spherical
to oval and distend the bacterial cells when they form within
them (­Figure 48-1). Clostridium species are found in soil and the
intestinal tracts of a variety of different animal species, including
those of healthy dogs and cats. Their spores are very resistant to
disinfection and can persist in the environment, including within
hospitals, for years. Despite their similarities, many differences
exist between C. difficile and C. perfringens that relate to epide-
miology, interpretation of diagnostic test results, and treatment.
C. difficile is the most common cause of hospital and
antimicrobial-associated diarrhea in humans and has also FIGURE 48-1  Fecal smear from a dog with diarrhea showing spore-distended
been associated with nosocomial diarrhea in dogs and cats in ­clostridial rods, leading to a characteristic “safety pin” and “tennis racquet” appearance.

458

The importance of C. perfringens and C. difficile as causes
of diarrhea in dogs and cats has been controversial, because
they can be isolated from both diarrheic and healthy animals.
For example, C. perfringens can be isolated using culture from
the feces of more than 70% of both diarrheic and nondiarrheic
dogs.11,12 The prevalence of C. perfringens colonization of
healthy cats appears to be lower than that in dogs, with isola-
tion rates of 43% to 63%.13 Similarly, toxin production can be
detected in both healthy and diarrheic dogs and cats, although
most studies have shown a correlation between production of
certain clostridial toxins and diarrhea.11
Clinical Features
Signs and Their Pathogenesis
Both C. difficile and C. perfringens cause diarrhea by produc-
ing toxins. The diarrhea can be mild and self-limiting or severe,
acute, and hemorrhagic, with life-threatening consequences that
relate to dehydration and hypovolemic shock. Inappetence,
vomiting, fever, and abdominal pain have also been described
in cats with C. difficile-associated diarrhea.14 Vomiting can also
occur in dogs. Although clostridial diarrhea is often described
as a large bowel–type diarrhea, clinical manifestations of both
small and large bowel diarrhea have been well documented.
C. difficile can produce up to five toxins, the most well studied
being toxin A (TcdA), toxin B (TcdB), and C. difficile binary toxin
(CDT, an ADP-ribosyltransferase that is made up of two compo-
nents). CDT-positive strains have not been reported in dogs and
cats. In contrast to humans, a history of antibiotic use does not
always seem to be necessary for colonization and disease in dogs
and cats, although in one study administration of antimicrobials
before admission and the use of immunosuppressive drugs during
hospitalization were risk factors for nosocomial colonization.4
Toxins A and B are thought to be most important in the patho-
genesis of diarrhea.15 These are large toxins that inactivate the
Rho family of GTPases, which regulate intracellular actin dynam-
ics. The result is a disruption of the cell cytoskeleton, with subse-
quent rounding and death of intestinal epithelial cells, and loss of
tight junctions. The two toxins also cause release of inflammatory
cytokines from mast cells, macrophages, and epithelial cells.15
In dogs, C. perfringens infection has been associated with
nosocomial diarrhea,16 hemorrhagic enteritis,17-19 and acute
and chronic large bowel diarrhea. C. perfringens diarrhea in
dogs and cats is thought to occur subsequent to massive prolif-
eration and sporulation of resident clostridial organisms in the
intestinal lumen following an alteration in local intestinal condi-
tions. Decreased peristalsis, the effects of antimicrobial drugs on
the resident intestinal microflora, diet changes, and/or co-infec-
tions with other intestinal pathogens has the potential to trigger
sporulation. C. perfringens produces CPE on sporulation, but
other toxins and bacterial virulence factors may also be involved
in the pathogenesis of diarrhea, because diarrhea can occur in
the absence of CPE production. CPE is a pore-forming protein
toxin that is released when vegetative clostridial cells lyse and
release their spores. It binds primarily to claudin, a component
of epithelial tight junctions, which leads to increased paracellu-
lar permeability.20 The role of CPE in the development of diar-
rhea in dogs and cats is unclear, because CPE is detected in 34%
of diarrheic dogs, and in 5% to 14% of nondiarrheic dogs.11,12
There may be an association between the detection of CPE in
dogs with acute hemorrhagic diarrheal syndrome (AHDS),
as CPE was detected in 8 of 12 dogs (67%) with AHDS.18
CHAPTER 48  Enteric Clostridial Infections 459
FIGURE 48-2  Abdominal ultrasound image showing thickening and corrugation of
the small intestinal wall of a 2-year-old intact male chihuahua with Clostridium perfrin-
gens–associated vomiting, hematemesis, diarrhea, and hematochezia. Large numbers of
spore-forming gram-positive rods were seen on a fecal smear. C. perfringens enterotoxin
was detected in the feces using an ELISA assay.
The prevalence of C. perfringens–associated diarrhea in cats is
much lower than in dogs, and CPE has been detected at a simi-
lar rate in diarrheic and nondiarrheic cats (2% to 4.1%).13 In
another study, CPE was detected with ELISA in 19.6% of 46
diarrheic dogs versus 2.1% of 48 healthy dogs, and in 8.3%
of 48 diarrheic cats versus 0% of 39 healthy cats.21 In a PCR-
based study of cats and dogs in an animal shelter in Florida,
the C. perfringens alpha toxin gene was detected in 64% of 50
diarrheic dogs and 40% of 50 nondiarrheic dogs, which was
significantly different.22 In contrast, it was detected in 42% of
50 diarrheic and 50% of 50 nondiarrheic cats in the same shel-
ter.23 Thus the role that C. perfringens plays in feline diarrhea,
if any, remains unclear.
Physical Examination Findings
Physical examination findings in dogs and cats with severe clos-
tridial diarrhea include dehydration, fever, and abdominal pain,
with or without signs of hypovolemic shock. Rectal examination
may reveal loose or watery stools, sometimes with fresh blood
and mucus.
Diagnosis
Laboratory Abnormalities
Complete Blood Count and Chemistry Panel
Basic laboratory testing in dogs and cats with clostridial diarrhea
usually reveals mild and nonspecific changes. A neutrophilic leu-
kocytosis with a left shift and toxic changes may be present in
dogs and cats with acute clostridial gastroenteritis. In addition, a
discordant hematocrit and serum total protein concentration can
be identified in some dogs with C. perfringens and C. difficile-
associated diarrhea. The hematocrit can be markedly increased
in the face of a normal or low-normal total protein.
Diagnostic Imaging
Findings on plain abdominal radiography and abdominal
ultrasound examination in animals with clostridial diarrhea
are typically unremarkable or show fluid-filled intestinal
loops. Mild thickening and corrugation of the small intestinal
or colonic wall may be detected on ultrasound examination
(Figure 48-2).
460 SECTION 2  Bacterial Diseases
TABLE 48-1
Diagnostic Assays Available for Clostridial Enterocolitis in Dogs and Cats
Specimen
Assay Type Target
Bacterial Feces C. perfringens and C. difficile
­isolation
Common Feces C. difficile
­antigen test
Toxin immu- Feces C. perfringens enterotoxin
noassays (CPE); C. difficile toxin A
and toxin B
PCR Feces Clostridium toxin gene DNA
Microbiologic Testing
Diagnostic assays available for clostridial enterocolitis in dogs
and cats are shown in Table 48-1. Because organisms and their
toxins can be found in healthy and diarrheic animals, the results
must be interpreted in light of the history, clinical signs, and
diagnostic testing to rule out other causes of disease.
Microscopic Examination
Spore-forming rods may be identified on microscopic examina-
tion of fecal smears that have been stained with Wright stains
(see Figure 48-1). Rods that contain endospores have the appear-
ance of “tennis racquets” or “safety pins.” However, there is no
association between fecal endospore counts and the presence
of diarrhea, or between endospore counts and the detection of
CPE in fecal specimens. Detection of fecal endospores is thus an
unreliable test for diagnosis of C. perfringens diarrhea in dogs
and cats and should not be used as a stand-alone test to make
clinical inferences in diarrheic dogs and cats.11,24
Bacterial isolation.  Both C. difficile and C. perfringens can
be isolated from feces of dogs and cats using strict anaerobic
conditions for incubation. C. perfringens is more tolerant of
oxygen than C. difficile and therefore may be easier to isolate
(hence the name difficile, because of its difficulty to isolate in
the laboratory).
C. difficile can be isolated from 0% to 40% of diarrheic and
healthy dogs. When fecal culture for C. difficile is performed
on a viable fecal specimen by a reputable laboratory, a nega-
tive result suggests that C. difficile is not present. However, a
positive culture does not differentiate between toxigenic and
nontoxigenic strains, and further testing for toxin is warranted.
Because of the high carriage rate of C. perfringens in both
healthy and diarrheic dogs (>70% in both groups), routine
isolation of C. perfringens is not recommended for diagnosis,
unless epidemiologic studies that require genetic typing are
undertaken in outbreak situations. Quantitative stool spore
counts have been used to aid diagnosis of C. perfringens diar-
rhea in humans. The stool is treated with heat or alcohol after
which it is cultured anaerobically and colonies are counted.
Performance
Isolation from feces does not imply that Clostridium is the cause of
disease. Allows genotyping and subsequent antimicrobial suscep-
tibility testing if needed. False negatives may occur, especially
for C. difficile, which requires strict anaerobic conditions.
Excellent negative predictive value for C. difficile (i.e., a negative
test result suggests C. difficile is not present). A positive test
result does not imply that C. difficile is the cause of disease.
Positive test results may occur in dogs and cats that do not have
diarrhea; negative results do not rule out clostridial diarrhea, as
other toxins may be involved and CPE is relatively labile.
Rapid (within hours). Positive results from feces do not imply
that Clostridium is the cause of disease. Toxin gene PCR assays
may be useful for diagnosis in conjunction with positive ELISA
results for toxin production. Negative results may occur if other
toxin genes are important or if inhibitors of PCR are present.
Median spore counts of greater than 106 per gram of feces col-
lected within 48 hours after the onset of illness are suggestive
of infection.25 Such counts can still be detected in humans with
no signs of illness, and the method, when used without assess-
ment of toxin production, has not proven useful for diagnosis of
C. perfringens diarrhea in dogs.12
ELISA for C. difficile Antigen
C. difficile antigen ELISA is easy to perform, rapid, and highly
sensitive. The antigen test detects “common antigen” (glutamate
dehydrogenase; GDH) that is predominantly present in toxi-
genic and nontoxigenic C. difficile strains and a few uncommon
Clostridium spp. This test has the same limitations as culture in
terms of detection of nontoxigenic strains, although a negative
test for GDH can virtually rule out infection with C. difficile.
Assays for Toxin Production
Several commercial serologic assays are available for detec-
tion of C. difficile toxin A and toxin B in feces. Fecal swab
specimens generally do not contain a sufficient quantity of
stool for these assays. Even when performed on larger vol-
umes of stool, the sensitivities and specificities of these assays
vary considerably, and the etiologic predictive values are rela-
tively low for all tests. In other words, a positive result does
not prove that a clostridial infection is the cause of a dog or
cat’s diarrhea. Currently, assays based on ELISA methodology
have shown the best correlation in dogs between clostridial
toxin detection and the presence of diarrhea in dogs.11,16,24
Panels should include assays for both C. difficile toxin A and
toxin B, because TcdA-negative, TcdB-positive strains have
been documented in dogs, and production of both toxins is
not needed for disease causation.26 The reference standard in
human medicine for C. difficile toxin production is a cell cul-
ture cytotoxicity assay, which uses Vero cells and neutralizing
antibody to inhibit cytopathic effects.27,28 However, culture
followed by molecular detection of toxin genes within the iso-
lated strains (also known as “toxigenic culture”) appears to
be a more sensitive reference standard.28 Both of these assays

are time consuming and expensive, require specific techni-
cal expertise, and are of limited availability. The use of mul-
tiple assays in sequence has been advocated for diagnosis of
C. difficile infection in humans.29 Specifically, fecal isolation
of C. difficile or detection of common antigen should be fol-
lowed with ELISA testing for toxin A and toxin B. Detection
of toxin but failure to identify C. difficile should be inter-
preted with caution, considering the relatively high sensitivity
of organism or common antigen detection methods compared
to toxin ELISA.
Detection of C. perfringens enterotoxin in stool is the most
widely used diagnostic tool for C. perfringens infection in both
humans and animals. Two commercially available immunoas-
says are currently used in veterinary diagnostic laboratories: a
reverse passive latex agglutination assay (RPLA) and an ELISA.
The assays have not been validated in animals to date. In addi-
tion, the RPLA has been associated with false-positive results
when compared to several different ELISA methods.
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR assays are commercially available for detection
of C. perfringens and C. difficile toxin genes in the feces of dogs
and cats. It is important to recognize that the presence of clos-
tridial toxin genes within fecal specimens does not prove that
clostridia are the cause of an animal’s diarrhea, because they do
not prove that the organism is actively producing toxin within
the intestinal tract of a patient. Conversely, a negative PCR result
for clostridial toxin genes does not rule out clostridial diarrhea,
as clostridia may be present that possess other virulence factors,
and inhibitors of PCR are common in fecal specimens. Real-
time PCR assays for C. difficile toxin genes have been devel-
oped for diagnosis of C. difficile diarrhea in humans.30 Some
assays can detect mutations within the toxin gene that correlate
with increased toxin production. However, it is still not well
understood how well the results of these assays correlate with
clinical illness. Similarly, detection of the cpe gene has been used
to diagnose C. perfringens diarrhea in humans.31 In one study,
fecal specimens from diarrheic dogs were more likely to be posi-
tive for CPE as detected using ELISA and PCR for the cpe gene
(28%) when compared with nondiarrheic dogs (4%). Such a
combination of testing for the toxin (CPE) via ELISA and the
enterotoxin gene (cpe) via PCR is likely superior to either test
alone.12 At the time of writing, real-time PCR assays that pro-
vide quantitative information on toxin gene copy numbers are
available through some veterinary diagnostic laboratories. The
degree to which toxin gene copy numbers correlate with clinical
signs requires further study.
TABLE 48-2
Antimicrobials That Can Be Used to Treat Complicated Clostridial Diarrhea in Dogs and Cats*
Drug Dose (mg/kg) Route
Metronidazole 10 PO
Ampicillin 22 PO
Amoxicillin 22 PO
Tylosin 5 to 10 PO
Erythromycin 15 PO
*Antimicrobial treatment of uncomplicated clostridial diarrhea is not recommended.
CHAPTER 48  Enteric Clostridial Infections 461
Treatment and Prognosis
Animals with suspected clostridial enterocolitis that are sys-
temically ill (fever, hemorrhagic gastroenteritis, inflammatory
or toxic leukogram) merit appropriate antimicrobial therapy
(Table 48-2). There is no documented evidence for a benefit of
antimicrobial therapy in dogs with uncomplicated Clostridium-
associated diarrhea, and most of these animals can be managed
supportively to ensure adequate hydration status without anti-
microbial therapy. In fact, a prospective, blinded study that ran-
domized dogs with hemorrhagic gastroenteritis that showed no
signs of sepsis to two groups, one treated with clavulanic acid–
amoxicillin for 7 days and one treated with placebo, showed no
difference in mortality rate, dropout rate, duration of hospital-
ization, or severity of clinical signs, either on any individual day
or over the course of disease.32
Metronidazole is the drug of choice for treatment of com-
plicated C. difficile infections. Oral vancomycin (which is not
absorbed from the gastrointestinal tract) is occasionally used in
human medicine to treat metronidazole-resistant isolates. The
vast majority of C. perfringens isolates from dogs are sensitive
to metronidazole, ampicillin, amoxicillin, tylosin, and macrolides
such as erythromycin.33 Because of the infrequency of resistance
to these drugs, the lack of known breakpoints for veterinary
anaerobes, and the lack of relationship between positive stool cul-
tures and disease, antimicrobial susceptibilities are not performed
on a routine basis for these organisms when isolated from dogs
and cats. Most animals with complicated clostridial enterocolitis
can be successfully treated with antimicrobials for 5 to 7 days,
and appropriate treatment is generally associated with clinical
improvement within 24 to 36 hours.
Immunity and Vaccination
No vaccines are available to prevent clostridial diarrhea in dogs
and cats.
Public Health Aspects
C. difficile is increasingly recognized as an important cause
of nosocomial diarrhea and pseudomembranous colitis in
humans, especially the elderly, which is induced by antimicro-
bial treatment (especially ampicillin, amoxicillin, clindamycin,
cephalosporins, and fluoroquinolones)34 or disruption of the
normal gastrointestinal flora. Humans with C. difficile infec-
tion develop bloating, and often foul-smelling diarrhea with
abdominal pain, usually 5 to 10 days after starting antibiotics.
Interval (hours) Duration (days)
12 5-7
8-12 5-7
12 5-7
24 5-7
8 5-7
462 SECTION 2  Bacterial Diseases
Other signs include fever, nausea, anorexia, and malaise. Rare
and life-threatening complications of pseudomembranous
colitis include acute toxic megacolon, an acute dilatation of
the colon with signs of obstruction, and colonic perforation.
Hypervirulent strains (such as PCR-ribotype 027) that produce
CDT, toxin A, and toxin B have been increasingly identified
recently that cause a more severe disease, higher relapse rates,
and increased mortality.35 Efforts are underway to develop vac-
cines for C. difficile infections in humans.36 There is evidence
that dogs may shed C. difficile strains similar to those that infect
humans. Human colonization and disease usually occurs in hos-
pital situations, and community-acquired infection is uncom-
mon, although concerns have been raised regarding the role of
CASE EXAMPLE
Signalment: “Bradley”, 2-year old male Chihuahua from
Grass Valley in northern California
History: Bradley was brought to an emergency clinic for
evaluation of a 12-hour history of hematemesis and
diarrhea. He initially vomited up dog food, after which he
continued to vomit hourly, and the vomiting progressed to
hematemesis. He also developed bloody diarrhea. He had
been inappetent and not drinking, and the owner felt he was
much quieter than usual. He primarily resided indoors and in
the owner’s backyard, and there was no history of travel or
access to toxins. There were two other apparently healthy
dogs in the household. He was up to date on vaccinations
for canine parvovirus, distemper virus, canine adenovirus,
and rabies. His diet consisted of commercial dry dog food,
dog bones, and table scraps.
Physical Examination:
Body Weight: 3.4 kg
General: Quiet, alert and responsive, ambulatory, 5% to 7%
dehydrated. Recumbent. T = 100.0°F (37.8°C), HR = 100
beats/min, RR = 30 breaths/min, mucous membranes pink
and tacky, CRT = 2 s.
Integument: A full coat with a small amount of flea excrement
was present. Bloody diarrhea contaminated the hair in the
perianal region.
Eyes, Ears, Nose, and Throat: No abnormalities were noted.
Musculoskeletal: Body condition score was 4/9.
Cardiovascular: The femoral pulses were fair and synchronous.
No murmurs or arrhythmias were auscultated.
Respiratory: Normal breath sounds were present in all lung
fields.
Gastrointestinal and Genitourinary: The dog was
nonpainful on abdominal palpation, but defecated red,
jelly-like material several times in the room following the
examination. He also urinated a large amount of odiferous
urine during the examination. No abnormalities were
detected on rectal examination apart from the abnormal
stool color and consistency.
Lymph Nodes: All lymph nodes were within normal limits on
palpation.
meat and companion animal food products in transmission of
the organism.6,37
C. perfringens infections are associated with sporadic and anti-
microbial-associated diarrhea in humans and CPE is one of the most
commonly identified causes of food poisoning in the industrialized
world.38 Clinical signs of infection include foul-smelling, frothy
diarrhea, cramping and abdominal pain, and occasionally nausea
and vomiting, usually 7 to 15 hours after eating suspected food,
especially meat products that have been cooked with improper
cooling and reheating.39 Vegetative cells are then ingested, and this
can be followed by massive sporulation with toxin production in
the intestinal tract. The zoonotic potential of C. perfringens iso-
lates from dogs and cats requires further investigation.
Laboratory Findings:
CBC:
HCT 46% (40%-55%)
MCV 68 fL (65-75 fL)
MCHC 36.3 g/dL (33-36 g/dL)
WBC 11,030 cells/µL (6000-13,000 cells/µL)
Neutrophils 7280 cells/µL (3000-10,500 cells/µL)
Band neutrophils 1324 cells/µL
Lymphocytes 1655 cells/µL (1000-4000 cells/µL)
Monocytes 772 cells/µL (150-1200 cells/µL)
Eosinophils 0 cells/µL (0-1500 cells/µL)
Platelets 273,000/µL (150,000-400,000 platelets/µL).
Neutrophils and band neutrophils showed mild to moderate
toxic changes.
Serum Chemistry Profile:
Sodium 147 mmol/L (145-154 mmol/L)
Potassium 4.5 mmol/L (3.6-5.3 mmol/L)
Chloride 111 mmol/L (108-118 mmol/L)
Bicarbonate 29 mmol/L (16-26 mmol/L)
Phosphorus 3.9 mg/dL (3.0-6.2 mg/dL)
Calcium 9.6 mg/dL (9.7-11.5 mg/dl)
BUN 15 mg/dL (5-21 mg/dL)
Creatinine 0.4 mg/dL (0.3-1.2 mg/dL)
Glucose 123 mg/dL (64-123 mg/dL)
Total protein 5.1 g/dL (5.4-7.6 g/dL)
Albumin 3.1 g/dL (3.0-4.4 g/dL)
Globulin 2.0 g/dL (1.8-3.9 g/dL)
ALT 68 U/L (19-67 U/L)
AST 31 U/L (19-42 U/L)
ALP 30 U/L (21-170 U/L)
GGT < 3 U/L (0-6 U/L)
Cholesterol 168 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.042, pH 6.0, 1+ protein (SSA), 1+ bilirubin,
no hemoprotein, no glucose, 1-2 WBC/HPF, 0 RBC/HPF, rare
granular casts.
Coagulation Panel: PT 8.9 s (7.5-10.5 s), APTT 18.1 s (9-12 s);
fibrinogen 349 mg/dL (90-255 mg/dL).
Imaging Findings: Abdominal ultrasound: Mild mesenteric
and sublumbar lymphadenopathy was identified. The
small intestines had mild wall thickening and a corrugated
appearance (see Figure 48-2). The colon was fluid filled.

Fecal Examination: Fecal flotation: Negative for parasites.
Fecal sedimentation: Negative for parasites.
Microbiologic Testing: Parvovirus fecal antigen ELISA:
Negative.
Fecal direct smear: Large numbers of gram positive rods were
present, some with spores.
Fecal enteric panel: Clostridium difficile toxin A and toxin B anti-
gen ELISA negative. ELISA positive for Clostridium perfringens
enterotoxin. No Salmonella spp. cultured. No Clostridium dif-
ficile cultured. No Campylobacter spp. cultured.
Diagnosis: Suspected C. perfringens–associated gastroenteritis.
Treatment: Bradley was initially hospitalized and treated with
intravenous fluids, nothing by mouth, and metronidazole
SUGGESTED READINGS
Marks SL, Kather EJ, Kass PH, et  al. Genotypic and phenotypic
­characterization of Clostridium perfringens and Clostridium difficile
in diarrheic and healthy dogs. J Vet Intern Med. 2002;16(5):533-540.
Weese JS, Armstrong J. Outbreak of Clostridium difficile–associated
disease in a small animal veterinary teaching hospital. J Vet Intern
Med. 2003;17(6):813-816.
Weese JS, Staempfli HR, Prescott JF, et al. The roles of Clostridium dif-
ficile and enterotoxigenic Clostridium perfringens in diarrhea in dogs.
J Vet Intern Med. 2001;15(4):374-378.
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1. George RH, Symonds JM, Dimock F. Identification of C. difficile as
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2. Welch WH, Nuttall GHF. A gas-producing bacillus (Bacillus aero-
genes capsulatus, nov. spec.) capable of rapid development in blood
vessels after death. Bull Johns Hopkins Hosp. 1892;3:81-91.
3. Hobbs BC, Smith ME, Oakley CL, et al. Clostridium welchii food
poisoning. J Hyg. 1953;51(1):75-101.
4. Weese JS, Armstrong J. Outbreak of Clostridium difficile–­
associated disease in a small animal veterinary teaching hospital. J
Vet Intern Med. 2003;17(6):813-816.
5. Clooten J, Kruth S, Arroyo L, et al. Prevalence and risk factors for
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an intensive care unit. Vet Microbiol. 2008;129(1-2):209-214.
6. Weese JS, Finley R, Reid-Smith RJ, et al. Evaluation of Clostridium
difficile in dogs and the household environment. Epidemiol Infect.
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7. Lefebvre SL, Reid-Smith RJ, Waltner-Toews D, et al. Incidence of
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bution of Clostridium difficile PCR ribotypes in cats and dogs from
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11. Weese JS, Staempfli HR, Prescott JF, et al. The roles of Clostridium
difficile and enterotoxigenic Clostridium perfringens in diarrhea in
dogs. J Vet Intern Med. 2001;15(4):374-378.
12. Marks SL, Kather EJ, Kass PH, et  al. Genotypic and pheno-
typic characterization of Clostridium perfringens and Clos-
tridium difficile in diarrheic and healthy dogs. J Vet Intern Med.
2002;16(5):533-540.
CHAPTER 48  Enteric Clostridial Infections 463
(7.5 mg/kg IV q12h). Vomiting did not continue, but red
gelatinous diarrhea continued every hour for the next
12 hours. Within 24 hours, he had become bright and alert
and was offered a bland diet, which he ate well. He was
discharged and treated with metronidazole for an additional
5 days, with gradual reintroduction of his regular food.
Comments: Infection by C. perfringens was strongly suspected
in this case based on the combination of consistent
clinical signs, negative assay results for other pathogens, a
positive toxin ELISA assay, and response to treatment with
metronidazole. However, because the feces of healthy dogs
can contain C. perfringens enterotoxin, a definitive diagnosis
of C. perfringens enterocolitis could not be made.
13. Marks SL, unpublished observations, 2011.
14. Weese JS, Weese HE, Bourdeau TL, et  al. Suspected Clostridium
difficile–associated diarrhea in two cats. J Am Vet Med Assoc.
2001;218(9):1436-1439,1421.
15. Carter GP, Rood JI, Lyras D. The role of toxin A and toxin B in
Clostridium difficile–associated disease: past and present perspec-
tives. Gut Microbes. 2010;1(1):58-64.
16. Kruth SA, Prescott JF, Welch MK, et al. Nosocomial diarrhea asso-
ciated with enterotoxigenic Clostridium perfringens infection in
dogs. J Am Vet Med Assoc. 1989;195(3):331-334.
17. Sasaki J, Goryo M, Asahina M, et al. Hemorrhagic enteritis associ-
ated with Clostridium perfringens type A in a dog. J Vet Med Sci.
1999;61(2):175-177.
18. Cave NJ, Marks SL, Kass PH, et al. Evaluation of a routine diag-
nostic fecal enteric panel for dogs with diarrhea. J Am Vet Med
Assoc. 2002;221:52-59.
19. Schlegel BJ, Van Dreumel T, Slavić D, et al. Clostridium ­perfringens
type A fatal acute hemorrhagic gastroenteritis in a dog. Can Vet J.
2012;53:555-557.
20. McClane BA. The complex interactions between Clostridium
perfringens enterotoxin and epithelial tight junctions. Toxicon.
2001;39(11):1781-1791.
21. Leutenegger CM, Marks SL, Robertson J. Toxin quantification of
Clostridium perfringens is a predictor for diarrhea in dogs and cats
[Abstract]. J Vet Intern Med. 2012;26:794.
22. Tupler T, Levy JK, Sabshin SJ, et al. Enteropathogens identified in
dogs entering a Florida animal shelter with normal feces or diar-
rhea. J Am Vet Med Assoc. 2012;241:338-343.
23. Sabshin SJ, Levy JK, Tupler R, et al. Enteropathogens identified in
cats entering a Florida animal shelter with normal feces or diarrhea.
J Am Vet Med Assoc. 2012;241:331-337.
24. Marks SL, Melli A, Kass PH, et al. Evaluation of methods to diag-
nose Clostridium perfringens–associated diarrhea in dogs. J Am
Vet Med Assoc. 1999;214(3):357-360.
25. Birkhead G, Vogt RL, Heun EM, et al. Characterization of an out-
break of Clostridium perfringens food poisoning by quantitative
fecal culture and fecal enterotoxin measurement. J Clin Microbiol.
1988;26(3):471-474.
26. Kuehne SA, Cartman ST, Heap JT, et  al. The role of toxin
A and toxin B in Clostridium difficile infection. Nature.
2010;467(7316):711-713.
27. Meer RR, Songer JG, Park DL. Human disease associated with
Clostridium perfringens enterotoxin. Rev Environ Contam Toxi-
col. 1997;150:75-94.
28. Crobach MJ, Dekkers OM, Wilcox MH, et  al. European Society
of Clinical Microbiology and Infectious Diseases (ESCMID): data
review and recommendations for diagnosing Clostridium difficile-
infection (CDI). Clin Microbiol Infect. 2009;15(12):1053-1066.
464 SECTION 2  Bacterial Diseases
29. Schmidt ML, Gilligan PH. Clostridium difficile testing algorithms:
what is practical and feasible? Anaerobe. 2009;15(6):270-273.
30. Sloan LM, Duresko BJ, Gustafson DR, et al. Comparison of real-
time PCR for detection of the tcdC gene with four toxin immuno-
assays and culture in diagnosis of Clostridium difficile infection.
J Clin Microbiol. 2008;46(6):1996-2001.
31. Loh JP, Liu YC, Chew SW, et al. The rapid identification of Clos-
tridium perfringens as the possible aetiology of a diarrhoeal out-
break using PCR. Epidemiol Infect. 2008;136(8):1142-1146.
32. Unterer S, Strohmeyer K, Kruse BD, et  al. Treatment of aseptic
dogs with hemorrhagic gastroenteritis with amoxicillin/clavulanic
acid: a prospective blinded study. J Vet Intern Med. 2011;25(5):
973-979.
33. Marks SL, Kather EJ. Antimicrobial susceptibilities of canine Clos-
tridium difficile and Clostridium perfringens isolates to commonly
utilized antimicrobial drugs. Vet Microbiol. 2003;94(1):39-45.
34. Pépin J, Saheb N, Coulombe MA, et  al. Emergence of fluoroqui-
nolones as the predominant risk factor for Clostridium difficile–
associated diarrhea: a cohort study during an epidemic in Quebec.
Clin Infect Dis. 2005;41(9):1254-1260.
35. Cartman ST, Heap JT, Kuehne SA, et  al. The emergence of
“hypervirulence” in Clostridium difficile. Int J Med Microbiol.
2010;300(6):387-395.
36. Lee BY, Popovich MJ, Tian Y, et al. The potential value of Clos-
tridium difficile vaccine: an economic computer simulation model.
Vaccine. 2010;28(32):5245-5253.
37. Gould LH, Limbago B. Clostridium difficile in food and domes-
tic animals: a new foodborne pathogen? Clin Infect Dis.
2010;51(5):577-582.
38. Brynestad S, Granum PE. Clostridium perfringens and food-borne
infections. Int J Food Microbiol. 2002;74(3):195-202.
39. Shandera WX, Tacket CO, Blake PA. Food poisoning due
to Clostridium perfringens in the United States. J Infect Dis.
1983;147(1):167-170.
CHAPTER 49
Gastric Helicobacter-like Infections
Jane E. Sykes and Stanley L. Marks
Overview of Helicobacter Infections in Dogs
and Cats
First Described: 1881 by Rappin (France),1 who observed
­spiral bacteria in a dog’s stomach; the association
between H. pylori infection and disease in humans was
made in 1983 by Warren and Marshall (Australia).2
Cause: Various species of non–H. pylori helicobacters, rarely
H. pylori in cats (gram-negative spiral-shaped bacteria
that belong to the family Helicobacteraceae)
Primary Mode of Transmission: Fecal-oral and oral-oral trans-
mission proposed, possibly water-borne. Transmission
through contact with vomitus may also occur.
Affected Hosts: Humans and a variety of other animals are
colonized by helicobacters; an association with disease is
clearest for humans and ferrets
Geographic Distribution: Worldwide
Major Clinical Signs (Dogs and Cats): Possibly chronic vomiting
(gastric helicobacters) or diarrhea (intestinal helicobacters),
although evidence for a causative role in disease is weak
Differential Diagnoses: Dietary indiscretion, gastrointestinal
foreign body, chronic pancreatitis, inflammatory bowel dis-
ease, food-responsive enteropathy, eosinophilic fibrosing
gastritis (cats), bilious vomiting syndrome, gastrointestinal
neoplasia (lymphoma, mast cell neoplasia, gastric adeno-
carcinoma), chronic infiltrative infectious diseases of the
gastrointestinal tract (including feline infectious peritonitis,
mycobacteriosis, canine cryptococcosis, pythiosis), hypo-
adrenocorticism, hyperthyroidism, toxins (including drugs),
gastric helminthiasis (e.g., P
­ hysaloptera and Ollulanus spp.)
Human Health Significance: Dogs and cats may be a source
of human infection with non–H. pylori helicobacters that
have been associated with gastritis, gastroduodenal
ulceration, and low-grade mucosa-associated lymphoid
tissue (MALT) lymphoma in humans.
Etiology and Epidemiology
Helicobacter spp. are flagellate, gram-negative, microaerophilic,
curved to spiral-shaped motile bacteria. They are grouped
into gastric, hepatic, and intestinal Helicobacter species, with
intestinal species residing primarily in the large intestine.3,4 In
humans, gastric Helicobacter spp. are an important cause of
gastritis and gastroduodenal ulceration and increase the risk
for development of gastric adenocarcinoma and lymphoma. In
contrast, the extent to which Helicobacter spp. cause disease
in dogs and cats is not fully understood, and newly discovered
organisms in dogs and cats have drawn most attention in regard
to their zoonotic potential.5
Helicobacter pylori, the type organism, is the most important
species infecting humans. H. pylori infection is extremely rare in
cats and has only been identified in one colony of cats to date.6
It has not been described in dogs. Dogs and cats are generally
infected with gastric non–H. pylori helicobacters (also referred
to as gastric helicobacter-like organisms, or GHLOs).5 These
are much larger than H. pylori (5 to 10 µm long versus 1.5 to
3 µm long for H. pylori), and species cannot be differentiated
from one another based on their light or electron microscopic
appearance alone (Figure 49-1). The nomenclature of non–H.
pylori helicobacters is complicated. Species identified in dogs
and cats are listed in Table 49-1.4,7-16 Before their genetic charac-
terization, non–H. pylori helicobacters were originally referred
to as ­“Gastrospirillum hominis” and later “Helicobacter heil-
mannii.” Subsequent analysis of multiple gene sequences from
a variety of “H. heilmannii” from dogs and cats has revealed
that “H. heilmannii” is not one but a group of organisms that
includes H. felis, H. bizzozeronii, H. salomonis, and an organ-
ism that has been confusingly named H. heilmannii. These
organisms have also been implicated in human disease, albeit
less commonly than H. pylori.5
Depending on the study, between 60% and 100% of dogs
and cats carry non–H. pylori helicobacters, and gastric Heli-
cobacter spp. can be found in apparently healthy animals and
dogs and cats without signs of vomiting.5,17-22 H. felis infection
has been associated with gastric pathology in dogs in some
studies23 but not others.24 Dogs and cats are probably colo-
nized shortly after birth, as a result of fecal-oral and oral-oral
­transmission.25 Transmission through contact with vomitus may
also occur. Water-borne transmission may play a role in spread of
H. pylori to humans.26 Shelter and colony dogs and cats may
have a higher prevalence of colonization, most likely due to the
close ­proximity of animals to one another. One Helicobacter
species may be able to suppress the presence of another, so that
dogs and cats are primarily colonized with a single species,
although in some animals, the simultaneous presence of mul-
tiple species has been found.27-30 The organisms have a remark-
able ability to survive the low pH of the stomach, which they
resist by living deep in the mucus glands of the stomach and
through production of the enzyme urease. The urease catalyzes
the hydrolysis of urea to carbon dioxide and ammonia, which
raises the pH of the organism’s milieu. In dogs and cats, the
organisms are also found within the canaliculi and the cyto-
plasm of parietal cells.25,30,31
465
466 SECTION 2  Bacterial Diseases

concurrently.31 Experimental and natural infection of dogs and

cats with non–H. pylori helicobacters has been associated with
chronic lymphoplasmacytic gastritis and lymphoid follicular
hyperplasia. Gastroduodenal ulceration and marked altera-
tions in the gastric acid secretory axis, as occur in humans,
have not been observed in dogs and cats.24,33,34 However, not
all animals infected with non–H. pylori helicobacters have his-
topathologic evidence of gastritis, and no correlation has been
observed between the severity of gastric pathology and the
degree of colonization by helicobacters. More severe gastritis
with marked lymphoid follicular hyperplasia and neutrophilic
inflammatory infiltrates has been reported in cats and acutely in
puppies experimentally infected with H. pylori.35,36 The puppies
developed gastrointestinal signs, including vomiting and loose
stool, shortly after inoculation. It is possible that the degree of
bacterial invasion and gastritis in dogs and cats varies with the
­ elicobacter species or even strain.30 In humans, gas-
infecting H
tric pathology has been associated with possession of a number
of pathogenicity genes by H. pylori, including cagA (cytotoxin-
associated gene A), vacA (vacuolating cytotoxin), and iceA
A B (induced by contact with epithelium).37 The degree of vacuolat-
FIGURE 49-1  Structure of gastric Helicobacter species. Note the terminal bunches of ing cytotoxin production can also vary among strains.38
flagellar filaments. A, Helicobacter pylori. B, Gastric helicobacter-like organism. In human patients, H. pylori infection has been well associ-
ated with gastric low-grade, B-cell, mucosa-associated lymphoid
tissue (MALT) lymphoma. Eradication of H. pylori achieves
complete remission in some patients with H. pylori–positive
TABLE 49-1 early-stage gastric MALT lymphoma.39 One study suggested
a possible association between Helicobacter spp. infection and
Helicobacter Species Identified in Dogs, Cats, and Humans4,7-16 gastric lymphoma in cats,40 but additional studies are required
to determine the role of Helicobacter spp. infection in feline gas-
Species Location Host tric disease.
H. bizzozeronii Stomach Dog, humans Non–H. pylori helicobacters have been detected in the hep-
H. salomonis Stomach Dog atobiliary system of cats with neutrophilic cholangitis41 and
H. cynogastricus Stomach Dog lymphocytic cholangitis,42 although this may reflect ascending
H. heilmannii Stomach Dog, cat, humans bacterial infection secondary to an underlying disease process.
H. felis Stomach Dog, cat, humans H. canis was detected in the liver of a 2-month-old puppy with
H. pametensis Stomach Cat multifocal necrotizing hepatitis that had acute weakness and
H. baculiformis Stomach Cat vomiting, and died within hours.43 H. canis was also detected in
H. pylori Stomach Cat a colony of Bengal cats with diarrhea, but its role as a causative
“Flexispira rappini” Stomach, intestine Dog, cat agent was uncertain.44 Abundant organisms that genetically
H. bilis Stomach, intestine Dog, cat resembled H. canis were detected microscopically throughout
H. cinaedi Intestine Dog, cat the large intestine of a 2-month-old kitten with severe diarrhea,
H. fennelliae Intestine Dog, cat vomiting, dehydration, weight loss, and inappetence.3 In one
“H. colifelis” Intestine Cat study, the presence of heavy colonic infection with enterohe-
H. marmotae Intestine Cat patic Helicobacter spp. infection in a group of laboratory dogs
H. canis Intestine, liver Dog, cat, humans was associated with the presence of mucosal atrophy and fibro-
sis, and a possible role of Helicobacter spp. infection in canine
inflammatory bowel disease was suggested.29

Clinical Features Diagnosis

Signs and Their Pathogenesis
The majority of dogs and cats infected with Helicobacter spp.
show no clinical signs. Helicobacter infection of some dogs
and cats has been hypothesized to cause chronic intermittent
vomiting, inappetence, pica, belching, weight loss, fever, and
polyphagia.31 Unfortunately, strong evidence that supports
an association with such disease in dogs and cats is lacking.
Clinical signs in some animals with biopsy-confirmed infec-
tion resolve following specific therapy for gastric helicobacter
infection,31-33 although the results of one study were difficult
to interpret because an elimination diet was administered
Microbiologic Testing
Clinical diagnosis of Helicobacter infection in dogs and cats has
most commonly been based on histopathology, cytology, rapid
urease testing, and, to a lesser extent, PCR on gastric biopsies
(Table 49-2). Other tests such as the urea breath and blood
tests, fecal PCR, and serologic tests are primarily used in human
patients and in the research arena.
Diagnosis Using Cytology and Histopathology
Spiral organisms can be readily detected in touch impression
smears of gastric tissue obtained following biopsy or necropsy,
 CHAPTER 49  Gastric Helicobacter-like Infections 467

TABLE 49-2
Diagnostic Assays Available for Gastric Helicobacter Infection in Dogs and Cats*
Assay Specimen Type Target Performance
Bacterial isolation Gastric biopsies Helicobacter Can take up to 10 days. Low sensitivity for non–H. pylori
organisms helicobacters, which can be difficult to culture. Patchy
distribution may also lead to false negatives. Allows
­typing and antimicrobial susceptibility testing.
Cytology Impression smears Helicobacter Rapid and sensitive, but false negatives can occur.
of ­gastric biopsies organisms Patchy distribution may also lead to false negatives.
or ­cytology brush Does not provide information on inflammatory or
­specimens architectural changes in underlying tissue.
Histopathology Gastric biopsies Helicobacter Less sensitive than cytologic examination. Patchy
organisms ­distribution may also lead to false negatives. Use
of silver stains increases sensitivity when organism
­numbers are low. ­Allows determination of associated
gastric ­histopathology.
Rapid urease Gastric biopsies Helicobacter Rapid and very sensitive. Patchy distribution and low
testing urease ­organism loads may lead to false negatives. Very rare
false positives with other urease-producing organisms.
PCR Gastric biopsies Helicobacter DNA Rapid (within hours); can be highly sensitive and
specific. Sequence analysis can determine the species
involved. Primarily used on a research basis.
Serology Serum Antibodies to Moderate sensitivity in dogs and cats. Results do not
Helicobacter spp. ­correlate well with gastric histopathology and do not
­always correlate with active infection. Primarily used
in research settings.
Urea breath and Breath, blood Radiolabeled carbon Requires specialized detection equipment. Although
blood testing dioxide derived from ­sensitive, false negatives may occur, such as following
the activity of acid suppression. In dogs and cats, has been primarily
Helicobacter urease used in research settings to date.

*For all tests, positive results do not imply that Helicobacter is the cause of disease.

after staining with Gram or Diff Quik stains (Figure 49-2). A
cytology brush can also be used to obtain a specimen during
endoscopy. Several biopsies or brush specimens may need to be
evaluated from different regions of the stomach (fundus, cardia,
antrum/pylorus) when the distribution of organisms is patchy.
When organism burdens are high, the organisms can be detected
using histopathology, which allows assessment of concurrent
gastric pathology (Figures 49-3 and 49-4). The use of silver
stains (such as Warthin-Starry stain or Steiner stain), Giemsa,
or toluidine blue stain, as well as immunostaining, dramatically
increases sensitivity for organism detection (see Figures 49-3, C,
and 49-4, C).45
Bacterial Isolation
Isolation of gastric helicobacters is difficult and has low sen-
sitivity. Growth typically requires incubation on selective
media in microaerobic conditions for 5 to 10 days. Isolation is
advantageous in research settings because it allows subsequent
identification of the organism using conventional biochemical
testing, whole-cell protein profiling, and DNA analysis.21,46
It also allows antimicrobial susceptibility testing, which
has recently been recommended before treating humans for
H. pylori infection because of the increasing prevalence of anti-
microbial resistance.47 Resistance to antimicrobials has also
been documented in non–H. pylori helicobacters isolated from
dogs and cats.48
Rapid Urease Testing
The rapid urease test involves incubating a gastric biopsy in a
urea broth that contains the pH indicator phenol red. If gastric
helicobacters are present, helicobacter urease breaks down the
urea; with the release of ammonia, a rise in pH and a color
change occur. The change in color can occur within 1 to 3 hours,
although the broth is generally incubated at room temperature
for 24 hours. The test is very sensitive, but false-negative results
can occur if the distribution within the stomach is patchy or if
organism loads are low. False-positive results have rarely been
reported when other urease-producing bacteria are present in
the stomach, such as Proteus spp.
Detection of Helicobacter DNA
PCR assays have been extensively used to detect Helicobacter
species in tissues on a research basis, including those from dogs
and cats. Sequence analysis of the PCR products can allow iden-
tification of the Helicobacter species present. Fluorescent in
situ hybridization (FISH) has also been used to detect, localize,
and identify Helicobacter species infection in tissues from dogs
and cats.4,32,40,49
468 SECTION 2  Bacterial Diseases
FIGURE 49-2  Spiral-shaped gastric Helicobacter-like organisms visible in a Diff Quik–stained smear (arrows) of a gastric biopsy from a 2-year-old male neutered Cavalier King Charles
spaniel with chronic vomiting and regurgitation for which no other cause was apparent (1000× magnification).
Serology
A variety of serum ELISA and immunoblotting assays have
been used in humans to detect antibodies and screen for
H. pylori infection, and some can be used to some extent to
monitor the success of therapy.50,51 Serologic assays have been
used on a research basis to noninvasively detect non–H. pylori
­helicobacter infection in dogs and cats, in some studies demon-
strating ­moderate sensitivity and good specificity.52-54
Urea Breath and Blood Testing
Diagnosis and monitoring of the extent of H. pylori infec-
tion in human patients can also be accomplished noninva-
sively using urea breath and blood testing, which involves oral
administration of urea containing 14C or the stable isotope 13C.
After ingestion, the labeled urea is converted to ammonia by
­Helicobacter urease, and the released carbon is absorbed sys-
temically, where it can be measured in the blood. Subsequently,
the labeled carbon dioxide is exhaled and can be measured
in the breath. Acid suppression interferes with test results by
decreasing H
­ elicobacter urease activity, and so testing is gener-
ally performed several days after discontinuing treatment. False
positives can also occur.55 Urea breath and blood testing has
been used with success in dogs,56 and breath testing has been
used in cats20,57 both before and following specific treatment for
Helicobacter infection.
Stool Antigen Assays
Assays that detect H. pylori antigen in feces have been used in
human patients,55 but their use in dogs and cats has not been
described.
Pathologic Findings
Infection with non–H. pylori helicobacters in dogs and cats has
primarily been associated with chronic lymphocytic or lym-
phoplasmacytic gastritis, with lymphoid follicular hyperplasia
(see Figures 49-3, A, and 49-4, A), although the extent of
inflammation varies dramatically from absent to severe.19,58,59
An eosinophilic inflammatory component has been described in
some cats.58 Neutrophil and eosinophil infiltrates can accom-
pany mononuclear inflammation in some cats infected with
H. pylori.58,60 Organisms may be found in the ­superficial
mucus and crypts, and sometimes within parietal cells see
Figures 49-3 and 49-4).
Histopathologic findings in a cat infected with an intestinal
helicobacter included large numbers of densely packed spiral
bacteria covering the intestinal mucosa and present within the
crypts, and minimal inflammatory changes. Hepatic changes
in a dog with H. canis infection consisted of randomly distrib-
uted and coalescing hepatocellular necrosis with associated
­neutrophilic and mononuclear infiltrates.43
Treatment and Prognosis
Whether antimicrobial treatment should be used for
­Helicobacter infections in dogs and cats is unknown.
Humans with H. pylori infections are typically treated with
a ­combination of a proton pump inhibitor, amoxicillin, and
clarithromycin, with or without bismuth subsalicylate. Treat-
ment of humans is reserved for symptomatic individuals; the
development of antimicrobial resistance by H. pylori is increas-
ingly of concern.61 Treatment should be reserved for dogs and
cats with gastrointestinal signs that have no other identifiable
cause of illness, after a diagnosis of gastritis and Helicobacter
infection has been confirmed using biopsy. A 2-week course
of combination therapy with ­antimicrobials and a proton
pump inhibitor or an H2 antagonist does not reliably eliminate
gastric helicobacters in dogs and cats, although clinical signs
often resolve.33,56,62,63 Reinfection can occur in some animals
after treatment. This theory is underscored by a study of 20
dogs that were naturally infected with Helicobacter spp. and
treated with triple therapy ­(clarithromycin, amoxicillin, and
lansoprazole) for 7 days. The dogs were then randomized into
a control group kept in isolation, and an experimental group,
which was placed in contact with Helicobacter-positive dogs
for 60 days. Triple therapy was effective in 100% of the dogs;
however, recurrence of infection occurred in 80% of dogs in
 CHAPTER 49  Gastric Helicobacter-like Infections 469

A A

B B

C C
FIGURE 49-3  Histopathology of the stomach of a 2-year-old female spayed Mal- FIGURE 49-4  Histopathology of the stomach of a 14-year-old male neutered
tese terrier with chronic vomiting and regurgitation. A, Lymphoid follicular nodules. The domestic shorthair that was infected with FIV and was euthanized because of squamous
remaining lamina propria is infiltrated by small numbers of eosinophils, neutrophils, lym- cell carcinoma. Gastrointestinal signs were not reported. A, Severe, chronic, lymphofol-
phocytes, and plasma cells. H&E stain, 40× magnification. B, Large numbers of spiral- licular gastritis. H&E stain, 40× magnification. B, Abundant fine, spiral-shaped bacteria
shaped bacteria are visible in the superficial mucous and gastric glands. H&E stain, 1000× were visualized in the superficial mucous and within parietal cells (arrows). H&E stain.
magnification. C, Agyrophilic intralesional spiral-shaped bacteria within the gastric C, Warthin-Starry stain showing intracellular agyrophilic spiral-shaped bacteria (arrows);
glands. Warthin-Starry stain, 1000× magnification. 1000x magnification.
470 SECTION 2  Bacterial Diseases

TABLE 49-3
Antimicrobials Used with Success to Treat Helicobacter spp.
Infection in Dogs and Cats
Dose Interval Duration
Drug (mg/kg) Route (hours) (days)
Metronidazole 10-15 PO 12 14-21
Amoxicillin 22 PO 12 14-21
Bismuth 0.22 mL/kg PO 6-8 14-21
subsalicylate
Clarithromycin 5-10 PO 12 14-21
Omeprazole 0.7-1.0 PO 24 14-21
Famotidine 0.5-1.0 PO 12 14-21

The first three drugs are administered as a combination. Alternatively, clar-

ithromycin can be combined with amoxicillin and administered with either
omeprazole or famotidine. Bismuth compounds should be avoided in cats.
Omeprazole should be given on an empty stomach 30 minutes before a meal.
the experimental group, and in none of the dogs in the con-
trol group after 60 days.64 A 3-week course of amoxicillin,
metronidazole, and bismuth subsalicylate (Table 49-3) cleared
gastric helicobacters in dogs and cats, with resolution of vomit-
ing, but histopathologic evidence of gastritis did not resolve.32
Metronidazole could be substituted with clarithromycin.21 One
study showed that concurrent use of an H2 antagonist in dogs
did not change outcome.33 This is consistent with the lack of
abnormalities in the gastric acid secretory axis in dogs infected
with non–H. pylori helicobacters.24,34
Immunity and Vaccination
Gastric helicobacters persist in the stomach despite a vigorous
humoral immune response. Research has been underway
to develop an effective vaccine for H. pylori infection in
humans, and a variety of H. pylori antigens induce protection
in healthy human volunteers or mouse models.65 No vaccine
is available for Helicobacter infection in dogs and cats, and
a greater understanding of the role of gastric helicobacters in
canine and feline disease will be required in order to assess
the need for effective vaccines against these organisms.
Public Health Aspects
At least 50% of the world’s human population is infected with
H. pylori, although the prevalence of infection has decreased
in developed countries.66 In the Western world, 1% to 10% of
people develop gastroduodenal ulceration as a result of chronic
H. pylori infection. A smaller percentage of infected people
(<3%) develop gastric adenocarcinoma or MALT lymphoma
in association with infection. Duodenal ulceration results from
increased gastrin release in response to H. pylori–induced
­gastric antral inflammation. Factors that influence outcome are
not well understood, but an individual’s immune response to
FIGURE 49-5  Endoscopic image of lymphoid follicular gastritis in a 2-year-old
­maltese terrier with chronic vomiting and regurgitation that was associated with the
presence of large numbers of gastric helicobacter like-organisms. Note the ‘goose pimple’
appearance of the mucosa.
the organism, as well as the possession of virulence factors by
H. pylori strains, appears to play an important role.66 Eradication
of infection using antimicrobial therapy can cure gastroduode-
nal ulceration and MALT lymphoma, but gastric adenocarci-
noma generally persists despite treatment.67 Although H. pylori
has been documented to infect a colony of cats, infection of cats
with this species is extremely rare, and exposure to pets is not a
risk factor for H. pylori infection in humans.
Non–H. pylori gastric helicobacter infection is uncom-
mon in humans compared with H. pylori infection. Never-
theless, these helicobacters have also been associated with
chronic active gastritis, gastroduodenal ulceration, and
MALT ­lymphomas in people. Disease is generally less severe
than that caused by H. pylori.5 Clinical signs in humans may
be absent or consist of nausea, epigastric pain, vomiting,
hematemesis, heartburn, and decreased appetite. Treatment
with triple antibiotic therapy and bismuth subsalicylate can
resolve infection and gastritis. Pets have been suggested as
a source of these helicobacters for humans,5,7,68-71 and close
contact with dogs and cats is a risk factor for infection.72
Licking has been suggested as a mode of transmission, based
on the presence of non–H. pylori helicobacter DNA in the
oral cavity of dogs.25 Pigs play an even more important role
in zoonotic ­transmission of non–H. pylori gastric helicobac-
ters, the swine helicobacter H. suis being the most prevalent
­species ­identified in humans.5
Intestinal helicobacters found in dogs and cats, especially
H. cinaedi, have been associated with enteric disease and
­bacteremia in immunocompromised humans.73 The relative role
of dogs and cats in transmission of these organisms to humans
is unknown. Nosocomial spread of H. cinaedi was suggested
among immunocompromised humans in one report.74
 CHAPTER 49  Gastric Helicobacter-like Infections
CASE EXAMPLE
Signalment: “Mochi,” a 2-year-old female spayed Maltese
terrier from Dixon, CA
History: Mochi had been vomiting and regurgitating
intermittently since she was 1 year of age. As a puppy,
the owner (a veterinary student) reported intermittent
episodes of “burping.” The vomiting occurred 2 to 5 times
throughout the day and was not associated with eating,
drinking, or exercise. The vomitus usually consisted of brown
fluid with no recognizable food particles. Dietary changes
that included a digestible elimination diet containing a
novel, single protein source followed by a fat-restricted
prescription diet had not resulted in improvement in her
clinical signs. Therapy with cisapride (3 mg PO q8h) and
famotidine (2.5 mg PO q24h) or omeprazole (3.5 mg PO
q24h) resulted in only slight improvement in the frequency
of vomiting. Her appetite was appropriate, and there had
been no weight loss or diarrhea. She primarily lived indoors
with one cat. She was up to date on vaccinations, which
included those for rabies, distemper, parvovirus, and
canine adenovirus.
Physical Examination:
Body Weight: 3 kg
General: Bright, alert and responsive, hydrated, nervous.
Ambulatory on all 4 limbs. T = 100.4°F (38.0°C), HR = 120
beats/min, RR = 28 breaths/min, mucous membranes pink
and moist, CRT = 1 s.
Integument, Eyes, Ears, Nose, and Throat: No clinically
significant abnormalities were noted.
Musculoskeletal: The dog’s body condition score was 4/9 with
symmetrical muscling.
Cardiovascular, Respiratory, and Lymph Nodes: No
clinically significant abnormalities were noted.
Gastrointestinal and Genitourinary: Abdominal palpation
was unremarkable. “Burping” behavior was observed in
the room, which occurred without any prodromal signs
or abdominal effort. Lip-smacking behavior occurred
immediately afterwards. Rectal examination was normal.
Laboratory Findings:
CBC:
HCT 46.5% (40%-55%)
MCV 64.4 fL (65-75 fL)
MCHC 35.9 g/dL (33-36 g/dL)
WBC 5700 cells/µL (6000-13,000 cells/µL)
Neutrophils 3112 cells/µL (3000-10,500 cells/µL)
Lymphocytes 1830 cells/µL (1000-4000 cells/µL)
Monocytes 257 cells/µL (150-1200 cells/µL)
Eosinophils 479 cells/µL (0-1500 cells/µL)
Platelets 277,000/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 147 mmol/L (145-154 mmol/L)
Potassium 4.4 mmol/L (3.6-5.3 mmol/L)
Chloride 111 mmol/L (108-118 mmol/L)
Bicarbonate 23 mmol/L (16-26 mmol/L)
Phosphorus 5.3 mg/dL (3.0-6.2 mg/dL)
Calcium 10.6 mg/dL (9.7-11.5 mg/dl)
471
BUN 22 mg/dL (5-21 mg/dL)
Creatinine 1.1 mg/dL (0.3-1.2 mg/dL)
Glucose 95 mg/dL (64-123 mg/dL)
Total protein 5.7 g/dL (5.4-7.6 g/dL)
Albumin 4.0 g/dL (3.0-4.4 g/dL)
Globulin 1.7 g/dL (1.8-3.9 g/dL)
ALT 42 U/L (19-67 U/L)
AST 45 U/L (19-42 U/L)
ALP 52 U/L (21-170 U/L)
GGT < 3 U/L (0-6 U/L)
Cholesterol 241 mg/dL (135-361 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.040, pH 7.0, no protein, glucose, or ketones,
trace hemoprotein, 0-1 WBC/HPF, 0-1 RBC/HPF, moderate
lipid droplets.
Serum Pancreatic Lipase Immunoreactivity: <30 µg/L
(reference range 0-200 µg/L)
Imaging Findings:
Abdominal Radiographs: A moderate amount of gas was
identified within multiple small intestinal loops, but an
obstructive pattern was not identified.
Thoracic Radiographs: The cardiopulmonary structures were
within normal limits. A small amount of gas was visualized
within the esophagus on the left lateral projections.
Esophagram: Unremarkable study.
Abdominal Ultrasound: No abnormalities were identified.
Gastroduodenoscopy: Gross findings were suggestive
of diffuse lymphoid follicular hyperplasia, with a mild
“goose pimple” appearance to the gastric mucosa
(Figure 49-5). Multiple biopsies of all regions of the stomach
and multiple duodenal biopsies were obtained and
submitted for histopathology.
Histopathology: Stomach: Moderate, lymphofollicular,
eosinophilic, and lymphoplasmacytic gastritis with intralesional
spiral bacteria (see Figure 49-3). Duodenum: Mild, chronic,
lymphofollicular, eosinophilic, and lymphoplasmacytic
duodenitis and ileitis. Jejunum: Diffuse, mild, eosinophilic, and
lymphoplasmacytic enteritis.
Diagnosis: Chronic gastritis associated with Helicobacter
spp. infection; mild eosinophilic and lymphoplasmacytic
enteritis.
Treatment: Treatment for the Helicobacter infection was
initiated with metronidazole (30 mg PO q12h), amoxicillin
(60 mg PO q12h), and bismuth subsalicylate for 2 weeks.
The owner reported almost complete resolution of the
vomiting following treatment, but signs returned after
treatment was discontinued, and an additional course of
treatment was instituted. Treatment with a fat-restricted
prescription diet was continued. The frequency of vomiting
then decreased to once a week.
Comments: Whether the Helicobacter spp. infection in this dog
contributed to clinical signs or was an incidental finding in
a dog with underlying enteritis was not clear. The failure to
respond to multiple diet changes, including an elimination
diet, and the dramatic response to triple therapy supported
a possible role for Helicobacter spp. infection, or a diagnosis of
antibiotic-responsive gastroenteropathy that was unrelated
to Helicobacter spp. infection.
472 SECTION 2  Bacterial Diseases
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Haesebrouck F, Pasmans F, Flahou B, et  al. Gastric helicobacters in
domestic animals and nonhuman primates and their significance for
human health. Clin Microbiol Rev. 2009;22(2):202-223.
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CHAPTER 50
Leptospirosis
Jane E. Sykes
Overview of Leptospirosis
First Described: Heidelberg, Germany, 1886 (Adolf Weil)1
Cause: Leptospira spp. (a spirochete)
Affected Hosts: Dogs, rarely cats, more than 150 other mam-
malian species
Geographic Distribution: Worldwide
Major Clinical Signs: Fever, lethargy, reluctance to move,
anorexia, polyuria and polydipsia, vomiting, diarrhea,
icterus, respiratory difficulty
Major Differential Diagnoses: Nephrotoxicoses (e.g., NSAIDs,
grapes and raisins, ethylene glycol), Lyme nephritis, acute
glomerulonephritides, bacterial pyelonephritis, acute
pancreatitis, canine monocytic ehrlichiosis, Rocky Moun-
tain Spotted Fever, bacterial sepsis, leishmaniosis
Human Health Significance: Zoonosis. Human infection results
from direct or indirect contact with contaminated urine. No
signs, a mild influenza-like illness, or severe illness with mul-
tiorgan failure (Weil’s disease) have the potential to occur.
Etiologic Agent and Epidemiology
Leptospirosis is a widespread zoonotic disease caused by systemic
infection by pathogenic spirochetes of the genus Leptospira. Lep-
tospira spp. are thin (0.1 µm in diameter), flexible, motile, spiral-
shaped bacteria that have a hook-shaped end (leptos = “thin,”
spira = “coiled”). A huge variety of different mammalian species
may be infected by leptospires. These hosts can shed the organ-
ism in their urine and contaminate the environment. Infection of
incidental hosts results in disease that varies in severity from a
mild febrile illness to severe multisystemic disease. Both dogs and
cats can develop leptospirosis, but cats appear to be relatively
resistant to development of clinical illness. As a result, leptospi-
rosis is uncommonly described in cats.2-5
Both pathogenic and saprophyte Leptospira species exist in
nature. Saprophytic species, such as Leptospira biflexa, live in
water and soil and do not infect animal hosts. Disease in dogs
is primarily caused by the pathogenic species Leptospira inter-
rogans and Leptospira kirschneri. Within the genus Leptospira,
more than 250 different serovars of pathogenic leptospires are
recognized, which are distinguished based on differences in their
lipopolysaccharide (LPS) O antigens. The serovars are grouped
into more than 20 antigenically related serogroups (Table 50-1).
At least 10 serovars have been associated with disease in dogs
worldwide. Each serovar is adapted to one or more wild or
domestic animal reservoir host species (see Table 50-1).
474
Recently, PCR-based DNA typing methods have been used
to identify Leptospira strains on the basis of their genotype,
instead of using the serovar classification method. Specific geno-
types appear to associate more strongly with particular reser-
voir hosts and disease manifestations than has been found for
serovars.6,7 Multiple different leptospiral serovars have been
isolated from single outbreaks of human leptospirosis associ-
ated with specific clinical manifestations (such as pulmonary
hemorrhage).8 Therefore, virulence factors other than outer sur-
face antigens (which define serovars) may more important deter-
minants of clinical manifestations of disease than the identity of
the infecting serovar.
Pathogenic Leptospira species do not replicate outside the host
and are readily inactivated in the environment when exposed to
excessive heat, ultraviolet irradiation, a variety of disinfectants,
and freezing conditions. However, when conditions are optimal,
pathogenic leptospires can survive in water and wet soil for weeks
to months.9 Dogs that drink from, swim, or wade in environmen-
tal water sources can become infected; exposure to wildlife or
farm animals and their urine is also a risk factor. In developing
countries, dogs may be infected as a result of access to sewage.10
Although the “classic” signalment for a dog with leptospirosis is
a large-breed, outdoor, intact male dog, dogs of any age, breed,
and sex may be affected. For example, geriatric, small-breed dogs
that live in urban or suburban areas and that contact rodents
or rodent urine can develop the disease. Indeed, over the past
decade, residence in urban areas has emerged as a risk factor for
Leptospira infection in dogs in some parts of North America,
possibly due to exposure to urban wildlife species.11 Cats may be
infected after ingestion of infected prey, so outdoor exposure and
hunting behavior may be risk factors for cats.2
Outbreaks of disease in dogs have been correlated with periods
of high annual rainfall and may occur approximately 3 months
after wet weather.12-14 The peak seasonal distribution in parts
of North America where freezing winters occur is late fall,11,15,16
but in more temperate regions, peak seasonal distribution occurs
after months of high rainfall (such as in the winter in northern
California).13 Leptospirosis is most prevalent in parts of the world
where there is higher annual rainfall and a warm climate, but the
presence of infected reservoir hosts also influences the geographic
distribution of leptospirosis. Within North America, dogs that live
in or travel to Hawaii, northern California, Oregon, Washington,
the upper Midwest and Midwest, parts of Texas, Colorado, and
the northeastern and mid-Atlantic coastal regions may be more
likely to develop infection.17 Leptospirosis also occurs in dogs in
the southeastern United States,18 and the disease has been widely
reported in dogs from Ontario in Canada.16 Numerous reports
describe leptospirosis in dogs from across Europe.19 Major dis-
ease foci among people include the Caribbean, Central and South
America, India, Southeast Asia, South America, and Oceania.

TABLE 50-1
Selected Serovars of Leptospira interrogans and Leptospira kirschneri that Infect Dogs *
Species Serogroup Serovar
Leptospira Icterohaemorrhagiae Icterohaemorrhagiae
interrogans Canicola Canicola
Pomona Pomona
Australis Bratislava
Sejroe ND
Ballum Ballum
Leptospira Grippotyphosa Grippotyphosa
kirschneri
*Isolated from dogs with naturally-occurring leptospirosis, or that induce disease in dogs after experimental inoculation.
Worldwide, the most common serovars thought to infect
dogs before the introduction of canine Leptospira vaccines
several decades ago were Icterohaemorrhagiae and Canicola.20
Since that time, widespread seroconversion to other serovars,
especially Grippotyphosa and Pomona in North America, has
been noted in sick dogs. Increased testing for these serovars may
have contributed to this phenomenon, as well as increased con-
tact between dogs and wildlife and farm animal reservoir hosts
for these serovars.21 Serovars that currently cause disease in the
dog population are poorly understood, because the results of
serology do not accurately predict the infecting serovar,22 and
culture and identification of leptospiral serovars is insensitive,
requires significant technical expertise, and is not widely avail-
able (see Diagnosis, later).23
Clinical Features
Signs and Their Pathogenesis
Pathogenic leptospires penetrate intact mucous membranes or
abraded skin. They multiply rapidly in the bloodstream as early
as 1 day postinfection, which may be followed by the develop-
ment of vasculitis and multiorgan failure. The spirochetes can
produce a variety of toxins, including phospholipases, sphingo-
myelinases, and pore-forming proteins. Infection follows direct
contact with infected urine or, more commonly, contact with
soil, water, food, or other fomites that have been contaminated
with infected urine. Transmission has also occurred as a result
of bite wound inoculation (such as from infected rodents),
ingestion of infected tissues, and venereal and placental transfer.
The incubation period for acute leptospirosis is approxi-
mately 7 days, but may be shorter or longer depending on the
dose and strain virulence of the leptospires involved, and the
host immune response.24 Dogs can also develop signs of chronic
kidney disease months or more than a year after acute kidney
injury. Early renal damage in this situation may be associated
with transient or undetected clinical signs, which are subse-
quently followed by either clearance or persistence of infection
and progression of renal damage. Longer incubation periods
might occur in cats,2 but further study is required.
Leptospirosis varies considerably in severity, which may con-
tribute to underrecognition of the disease. Many dogs probably
CHAPTER 50  Leptospirosis 475
Potential Reservoir Hosts Country
Rats United States, France
Dogs India, United States
Horses, cattle, sea lions United States
Pigs, hedgehogs, horses United States
Mice Germany
Mice, gray squirrels, muskrats, United States
opossums
Raccoons, fox squirrels, muskrats, United States
bobcats, voles, mice, rats, cattle,
horses
FIGURE 50-1  Uveitis in an Australian shepherd with leptospirosis with leakage of
serum bilirubin into the aqueous humor.
show no signs of illness or develop only a mild, transient febrile
illness. Severe clinical signs and death result from renal failure
and/or injury to the liver and lungs. Leptospirosis should be sus-
pected in dogs that develop signs of acute febrile illness, kidney
and hepatic injury, uveitis, or pulmonary hemorrhage; it should
also be suspected in breeding dogs with abortion, stillbirths, or
neonatal deaths.20 Clinical signs in affected cats resemble those in
dogs, although some cats have lacked evidence of hepatic injury.2
Fever occurs only at the onset of illness and is often accom-
panied by shivering, reluctance to move, and generalized muscle
tenderness that may result from myositis. This may be followed
by signs of lethargy, polyuria and polydipsia, inappetence, vom-
iting, and diarrhea.24-27 Gastrointestinal signs in some dogs may
result from pancreatitis and enteritis, in addition to acute renal
and liver damage. Polyuria and polydipsia may result from a
decreased responsiveness of the inner medullary connecting ducts
to antidiuretic hormone (acquired nephrogenic diabetes insipi-
dus). However, some dogs show decreased thirst and urination.
Liver involvement leads to icterus in some dogs (Figure 50-1).
Components of leptospiral endotoxin such as oleic and linoleic
acid are thought to interfere with hepatic transport pumps,
which leads to functional hepatic impairment.28 Hepatocyte
apoptosis and injury to hepatocyte membranes is also suspected
476 SECTION 2  Bacterial Diseases

TABLE 50-2
Complete Blood Count Findings at Admission in Dogs with Leptospirosis from Northern California*
Percent Below Percent Within Percent Above Range for
Reference the Reference the Reference the Reference Dogs with Number
Test Range Range Range Range Leptospirosis Tested
Hematocrit (%) 40-55 78 22 0 12-48 54
Neutrophils (cells/µL) 3000-10,500 0 35 65 3474-14,311 54
Band neutrophils (cells/ Rare 0 63 37 0-2954 54
µL)
Monocytes (cells/µL) 150-1200 0 65 35 162-4594 54
Lymphocytes (cells/µL) 1000-4000 30 70 0 162-3769 54
Eosinophils (cells/µL) 0-1500 0 100 0 0-970 54
Platelets (cells/µL) 150,000- 28 61 11 40,000-522,000 53
400,000

*Diagnosis based on results of acute and convalescent phase serology together with consistent clinical signs.

to occur; leptospirosis in humans and guinea pig models is asso-
ciated with loss of the cellular adhesion molecule E-cadherin on
hepatocytes.20
Less commonly, uveitis, myocardial damage, or reproductive
complications occur. Leptospiral DNA can be detected in the
aqueous humor of human patients with leptospiral uveitis, and
the development of uveitis may be delayed up to 18 months after
the onset of acute illness.29 ECG alterations can occur in dogs that
suggest myocardial damage.30 Abortion can occur during preg-
nancy.31 Stillbirth and neonatal deaths have also been described.
Bleeding tendencies in dogs with leptospirosis may be mani-
fested as petechial hemorrhages, hematuria, hematemesis, epi-
staxis, hematochezia, or melena.25,30,32 Although the precise
mechanisms are unknown, bleeding tendencies may reflect the
presence of endothelial damage and disordered coagulation. In
human patients, thrombocytopenia (≤100,000 platelets/µL) and
prolonged PT have been associated with bleeding.33,34 Tachy-
pnea may be noted as a result of severe pulmonary hemorrhage
syndrome (SPHS).35,36 SPHS is recognized commonly in humans
with severe leptospirosis, is not associated with significant lung
colonization by the spirochete, and seems to have an immune-
mediated pathogenesis.37 The prevalence of SPHS in dogs appears
to be particularly high in continental Europe. For example, pul-
monary complications occurred in more than two thirds of dogs
with leptospirosis in Berlin.36 Vasculitis can also contribute to the
development of peripheral edema and mild pleural and perito-
neal effusions. In human patients, aseptic meningitis is a common
complication of leptospirosis and results in signs of severe head-
ache, delirium, and less commonly seizures and altered sensory
perception.38 Whether meningitis occurs in dogs is unknown.
The extent to which leptospires can cause chronic hepatic
injury in dogs is also unknown. In one report from France, lepto-
spires were detected in bile canaliculi of young dogs with chronic
hepatitis using immunohistochemistry and electron microscopy.
These dogs showed signs of poor growth, weight loss, and asci-
tes.39,40 Attempts to detect Leptospira DNA in liver tissue from
dogs with chronic active hepatitis have been unrewarding.
Physical Examination Findings
Findings on physical examination of dogs with leptospirosis vary
with disease severity, chronicity, and the infecting leptospiral
strain. Severely affected dogs may show fever, dull mentation,
dehydration, mild generalized peripheral lymphadenopathy,
diffuse muscle pain, and pain on palpation of the abdomen
or kidneys. Fever is typically only present early in the course
of infection. In oliguric or anuric dogs that have been treated
aggressively with intravenous fluids, evidence of overhydration
may be present. Rarely, arrhythmias are detected on cardiac aus-
cultation. Careful ophthalmologic examination may reveal con-
junctivitis, scleral injection, and occasionally uveitis. Mild pallor,
petechial or ecchymotic hemorrhages, and, less commonly, epi-
staxis may be present. Respiratory difficulty, tachypnea, and
harsh lung sounds may be evident in dogs that develop pulmo-
nary complications. Dogs with liver involvement may be icteric.
Rectal examination occasionally reveals melena or hematochezia.
Diagnosis
Laboratory Abnormalities
Complete Blood Count
The hemogram in dogs with leptospirosis may reveal neutro-
philia, sometimes with bandemia; nonregenerative anemia;
lymphopenia; and/or thrombocytopenia (Table 50-2). Throm-
bocytopenia occurs in up to 53% of dogs.25,26,30,41 The mecha-
nism is unclear; disseminated intravascular coagulation (DIC)
or immune-mediated mechanisms may be involved.
Serum Biochemical Tests
Findings on the serum chemistry panel vary depending on lep-
tospiral strains that circulate in a particular geographic region
(Table 50-3). In all geographic locations, a combination of
azotemia and elevated liver enzyme activities, especially when
thrombocytopenia is present, should raise suspicion for leptospi-
rosis. Azotemia is generally present in more than 80% to 90%
of dogs,25,26,30,42 although in one European study, increased
serum creatinine concentration was present in only 57% of
affected dogs.41 Uncommonly, dogs exhibit increased liver
enzyme activities in the absence of azotemia. Signs of hepatic
dysfunction are associated with minimal histologic damage to
the liver and include variable, and generally mild to moder-
ate, increases in serum ALT, AST, and ALP activities and total
bilirubin concentration.
 CHAPTER 50  Leptospirosis 477

TABLE 50-3
Serum Biochemistry Findings at Admission in Dogs with Leptospirosis from Northern California*
Percent Below Percent Within Percent Above Range for
Test (Number of Reference the Reference the Reference the Reference Dogs with Number of
Dogs Tested) Range Range Range Range Leptospirosis Dogs Tested
Sodium (mmol/L) 145-154 33 61 6 134-162 54
Potassium (mmol/L) 4.1-5.3 35 44 20 2.8-7.6 54
Chloride (mmol/L) 105-116 57 37 6 98-118 54
Bicarbonate (mmol/L) 16-26 33 61 6 11-28 54
Calcium (mg/dL) 9.9-11.4 39 44 17 7.2-10.9 54
Phosphorus (mg/dL) 3.0-6.2 0 9 91 2.7-13.0 54
Creatinine (mg/dL) 0.5-1.6 0 0 100 0.4-3.3 54
BUN (mg/dL) 8-31 0 2 98 8-110 54
Albumin (g/dL) 2.9-4.2 96 4 0 0.9-3.8 54
Globulin (g/dL) 2.3-4.4 9 78 13 1.6-4.7 54
Cholesterol (mg/dL) 135-345 4 91 79-408 54
Total bilirubin (mg/ 0-0.4 0 69 32 0-3.1 54
dL)
ALT (U/L) 19-70 0 43 48 17-1443 54
ALP (U/L) 15-127 0 67 33 29-985 54
CK (U/L) 46-320 31 8 62 5-258,720 13

*Diagnosis based on results of acute and convalescent phase serology together with consistent clinical signs.

Other findings on the serum chemistry panel include
hypoalbuminemia and a variety of electrolyte abnormalities.
Hyponatremia, hypochloremia, or hyperphosphatemia occur
in most dogs. Decreased expression of sodium transporters
by proximal convoluted tubule cells contributes to impaired
sodium resorption, increased distal sodium delivery, and potas-
sium wasting.43 As a result, dogs with acute oliguric renal
failure may have paradoxical hypokalemia.
Moderately and occasionally markedly increased serum CK
activity is present in some dogs due to myositis. Increased serum
troponin concentrations have been found in some dogs with
leptospirosis, which suggests myocardial damage.30 Increased
serum pancreatic lipase immunoreactivity may occur, probably
as a result of pancreatitis.
Urinalysis
Findings on urinalysis in dogs with leptospirosis include isosthe-
nuria, occasionally hyposthenuria, and variable glucosuria, pro-
teinuria, pyuria, cylindruria, and bilirubinuria.20,26,30,42 Urine
protein to creatinine (UPC) ratios may be increased. Glucosuria
was present in 77% of 44 dogs with leptospirosis at the author’s
institution, and in 21 dogs that had UPC ratios measured, the
ratios ranged from 0.4 to 11.6 (median, 3, reference range
less than 0.5). Most proteins in the urine are of low molecular
weight, which supports a tubular rather than glomerular ori-
gin of these proteins.30,44 Leptospires are not visible in urine
sediment by routine light microscopy.
Coagulation Function
Aside from thrombocytopenia, analysis of hemostatic func-
tion in dogs with leptospirosis shows variable increases in
fibrinogen, D-dimer, and fibrinogen degradation product con-
centrations. Antithrombin activity may be decreased. Prolonga-
tions in PT and PTT have been present in up to 15% of tested
dogs.20,26,30 PT prolongations are associated with mortality in
human patients.34 In some dogs, a shortened PT is present,42
possibly because of DIC. In human patients, leptospirosis has
been associated with an imbalance between coagulation and
fibrinolysis.34
Diagnostic Imaging
Plain Radiography
Plain thoracic radiography in dogs with leptospirosis may show
no abnormalities, or mild to severe, diffuse interstitial pulmo-
nary patterns may be present. Thoracic radiographs from dogs
with SPHS can show severe nodular to diffuse interstitial and
alveolar patterns (Figure 50-2). Uncommonly, mild pleural
effusion is present.
Sonographic Findings
Findings on abdominal sonography include mild renomegaly,
increased renal cortical echogenicity, mild pyelectasia, perire-
nal fluid accumulation or mild ascites, and a medullary band
of increased echogenicity within the kidneys (Figure 50-3).45
None of these signs are specific for leptospirosis. Ultrasono-
graphic changes suggestive of pancreatitis are occasionally pres-
ent, such as enlargement and hypoechogenicity of the pancreas.
Thickening of the gastric, and less commonly small intestinal
wall may also be present. Mild to moderate hepatomegaly
and splenomegaly with mottling of the splenic echotexture
may be detected, with or without evidence of mild abdominal
lymphadenomegaly.
478 SECTION 2  Bacterial Diseases
A
B is slow, can be insensitive, and is not widely available. Special
C resis can also be performed.23 Despite the difficulties associated
FIGURE 50-2  Clinical images from a 4-year-old male neutered Havanese terrier with
severe leptospiral pulmonary hemorrhage syndrome and anuric renal failure. A, Right lat-
eral thoracic radiograph. A severe, diffuse alveolar pattern is present. Mechanical ventila-
tion was needed and ultimately euthanasia was performed because of respiratory failure.
Histopathology of a lung biopsy showed severe pulmonary hemorrhage (B) and marked
alveolar histiocytosis (C).
FIGURE 50-3  Abdominal sonogram image showing perirenal fluid accumulation in a
1.5-year-old male neutered kelpie with renal failure due to acute leptospirosis.
Microbiologic Tests
Diagnostic assays available for leptospirosis in dogs are
described in Table 50-4. Most often, diagnosis is based on acute
and convalescent phase serology.
Darkfield Microscopy
Examination of urine sediment using darkfield microscopy is
insensitive for diagnosis of leptospirosis. Considerable experi-
ence is required to accurately identify the spirochetes, so false
positives also have the potential to occur.
Culture
Culture of leptospires is not routinely performed, because it
leptospira growth media is required, such as Ellinghausen-
McCullough-Johnson-Harris (EMJH) medium. Cultures must
be incubated for up to 3 to 6 months, and overgrowth with
other bacteria can occur if cultures become contaminated.
Venous blood and/or urine should be collected using aseptic
technique before initiating antimicrobial drug treatment. Ide-
ally, a few drops of blood or urine are inoculated directly into
culture medium alongside the patient. In humans, blood is opti-
mally collected during the initial febrile period; urine can be
collected after the first week of illness. Because the exact time of
infection may be unknown, submission of both blood and urine
can increase the chance of a positive test result.
Only certain reference laboratories have expertise in culture
of leptospires and the ability to identify leptospires to the serovar
level, which is performed using a cross agglutinin absorption
test. Genetic typing methods such as pulsed field gel electropho-
with culture and identification of leptospires, a proper under-
standing of the epidemiology of leptospirosis depends on the
use of these methods.
Serologic Diagnosis
Currently the test of choice for diagnosis of leptospirosis is serol-
ogy using the microscopic agglutination test (MAT).20 In this
assay, the laboratory mixes serial dilutions of patient sera with
a panel of live leptospiral serovars. The laboratory then reports
the agglutinating antibody titer for each of the serovars tested,

TABLE 50-4
Diagnostic Assays Available for Leptospirosis in Dogs
Assay Specimen Type Target
Darkfield microscopy Urine Leptospira organisms
Culture Whole blood, urine Leptospires
Serology (MAT) Serum Antibodies against various
leptospiral serovars
Histopathology Kidney tissue collected Leptospires
via biopsy or
necropsy
PCR Blood, urine, tissue Leptospira DNA
specimens
which is the highest dilution of serum that causes the organisms
to agglutinate as assessed using darkfield microscopy. Seroposi-
tive dogs can have high titers to multiple serovars tested because
of serologic cross-reactivity. The highest titer does not necessar-
ily predict the infecting serovar.22 The serovar that produces the
highest titer may also change when serology is repeated over the
course of illness.20
Documentation of a fourfold or greater increase in titer to
at least one serovar over time is usually required for diagnosis
using the MAT, because titers are often low or negative in
the first few days of illness, and a single high titer can result
from previous exposure or a history of vaccination for lep-
tospirosis. Later in the course of illness, a fourfold decrease
in titer may also be appropriate for diagnosis. Tradition-
ally, convalescent titers are performed 2 to 4 weeks after the
acute titer, although seroconversion can sometimes occur as
early as 3 to 5 days after the initial titer is obtained. Nev-
ertheless, successive titers should be spaced at least 1 week
apart, and it is critically important to use the same labora-
tory for each set of titers.20 Antimicrobial treatment has the
potential to blunt a rise in titer, but dogs usually serocon-
vert despite antimicrobial treatment.42 Titers that result from
previous vaccination, previous exposure or chronic infec-
tion generally change by one dilution over time (e.g., from
1:400 to 1:800) or remain static. Titers persist for at least
1 year after natural infection, although they often decline by
4 months after vaccination.46 Nevertheless, postvaccinal titers
can persist for longer than a year and be maintained at high
CHAPTER 50  Leptospirosis 479
Performance
Low sensitivity and specificity. Requires
considerable technical expertise to interpret
correctly.
Low sensitivity. Special media and prolonged
incubation times required. Difficult and not
widely available. Antimicrobial therapy may
lead to false-negative results. Currently the
only method that permits accurate serovar
identification.
False negatives can occur early in the course
of illness or with immunosuppression. False
positives can occur with a history of vac-
cination or with previous exposure. Paired
titers performed at the same laboratory
generally required for diagnosis. Interlabo-
ratory variation in results may occur.
Organisms may be visualized with silver
stains, immunohistochemistry, or fluores-
cence in situ hybridization. Antimicrobial
therapy may lead to false-negative results.
Sensitivity and specificity unclear and may
vary between assays offered by different
laboratories. Antimicrobial therapy may
lead to negative PCR results.
levels (≥1600), especially if ongoing exposure to field strains
occurs. Thus, although single positive titers can raise suspicion
for the disease, they cannot be used to confirm the diagnosis
of leptospirosis. After immunization, paradoxical cross-reac-
tivity to serogroups other than those used in the vaccine can
occur,46 so titers to non-vaccinal serovars in vaccinated dogs
do not necessarily imply natural exposure to field strains.
False-negative titers have the potential to occur if the
infecting serovar (or a related serovar) is not included in the
panel of serovars used to perform the test. Assays for diagno-
sis of human leptospirosis generally include a larger panel of
serovars (>20) than those used routinely in veterinary diagnos-
tics (5 to 7 serovars). Assays should optimally include serovars
from serogroups that are known to circulate in the local dog
population, although this information is not often readily
available.
The MAT is widely available and relatively inexpensive,
but is complex to perform and interpret. Performance of the
test is hazardous to laboratory workers because live patho-
genic serovars are used, and the test is difficult to standardize.
Considerable interlaboratory variation in results can occur,
in terms of both the magnitude of the titers reported and the
pattern of seroreactivity to each serovar included in the test.47
The identity of serovars used in the assay must be verified peri-
odically to ensure that cross-contamination or culture dete-
rioration has not occurred. A leptospirosis proficiency testing
scheme is offered by the International Leptospirosis Society
(ILS) to assist laboratories in the maintenance of quality
480 SECTION 2  Bacterial Diseases

assurance for the MAT.48,49 Veterinarians should strive to use

laboratories that participate in this quality assurance scheme.
Other serologic assays under investigation for diagnosis of
leptospirosis include ELISA assays for IgG and IgM antibod-
ies that are directed against leptospiral surface antigens.50-53
The performance of such assays have the potential to vary
geographically based on the serogroups that are prevalent in
the region. In Europe, a lateral-flow assay that detects IgM
(Test-it, Royal Tropical Institute/Life Assay Diagnostics) is
available for diagnosis of acute canine leptospirosis.50 This
assay appeared to have high clinical sensitivity for diagno-
sis of leptospirosis in dogs in the Netherlands (100%) and
the West Indies (78%), with a low rate of false positives
in healthy dogs from the Netherlands, the West Indies, and
Mexico (<5%).

Molecular Diagnosis Using the Polymerase Chain Reaction

Several veterinary diagnostic laboratories in the United States
and Europe offer PCR assays for the detection of the DNA of
pathogenic leptospires in blood and/or urine. The sensitivity
and specificity of these assays in dogs with leptospirosis has
not been thoroughly assessed and have the potential to vary
geographically, because of differences in shedding patterns
for different serovars. Assay performance also is likely to vary
from one laboratory to another. PCR assays have the poten-
tial to be advantageous for diagnosis early in the course of ill-
ness, when serologic tests are negative, or in dogs that possess
residual antibody after vaccination. Specimens for PCR testing
are best obtained before initiation of antimicrobial drug treat-
ment, and both blood and urine should be submitted in order
to optimize sensitivity. Negative results do not rule out a diag-
nosis of leptospirosis. Because subclinically infected dogs can
shed leptospires, a positive PCR test result on urine may not
necessarily correlate with illness. Vaccination of healthy dogs
with inactivated Leptospira vaccines should not lead to posi-
tive PCR assay results,54 so a history of Leptospira vaccination
should not interfere with PCR diagnosis. Currently available
PCR assays do not provide information on the infecting serovar
or serogroup.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with leptospirosis include
jaundice, petechial and ecchymotic hemorrhages, and some-
times fibrin thrombi throughout the body. The lungs of dogs
with SPHS may be wet, mottled, heavy, and dark-pink to red
in color.36 Ascites and/or pleural or pericardial effusion may
be present, and the intestinal lumen may be filled with blood
or contain melena. The kidneys of dogs with acute disease may
be mildly enlarged, pale and sometimes mottled or petechiated;
they may also contain infarcts. Dogs with chronic renal injury
secondary to the infection can have kidneys that are shrunken
and irregular (Figure 50-4).
Histopathologic Findings
Infection of the kidneys by leptospires leads to an interstitial
nephritis, with mixed inflammatory infiltrates. Acute tubular
necrosis can also occur as a result of vasculitis and renal isch-
emia (Figure 50-5, A).36 Histopathologic changes in the liver
are often mild and include mild random hepatic necrosis, mild
neutrophilic periportal hepatitis, cholestasis, or liver plate dis-
array (Figure 50-6). Multifocal, random, neutrophilic, and
FIGURE 50-4  Shrunken, irregular kidneys from a 16-year-old male neutered Jack
Russell terrier. The dog was treated for severe acute leptospirosis at 12 years of age. At
that time, after six dialysis treatments, the dog was discharged from the hospital with a
creatinine of 4.2 mg/dL. Four years later, the dog died for reasons unrelated to renal injury
but had evidence of chronic renal injury at necropsy. (Image courtesy UC Davis Veterinary
Anatomic Pathology Service.)
necrotizing myocarditis can also occur; myonecrosis is occa-
sionally described in skeletal muscle. Lung pathology in dogs
with SPHS resembles that described in human patients and
consists of intra-alveolar hemorrhage, pneumocyte necrosis,
and hyaline membrane formation, in the absence of significant
inflammation.
Silver stains such as Warthin-Starry stain and immunohisto-
chemistry can be used to visualize bacteria within tissue speci-
mens (see Figure 50-5, B). Fluorescence in situ hybridization
has also been used to identify and localize leptospires within the
kidneys (see Figure 5-1).
Treatment and Prognosis
Antimicrobial Drugs
Penicillins or doxycycline are recommended for initial treat-
ment of humans and dogs with leptospirosis. Treatment
should be initiated as early as possible, before the results of
diagnostic assays become available. In hamster models, doxy-
cycline clears spirochetes from all tissues, including the kidney,
within 3 days of infection; ampicillin is less effective at clear-
ing organisms from the kidney.55 Based on current evidence,
the ACVIM Consensus Statement on leptospirosis in dogs
 CHAPTER 50  Leptospirosis 481

B bilirubin concentration were mildly increased, and the dog seroconverted with the highest
FIGURE 50-5  A, Histopathology of the kidney of a dog that died of leptospirosis.
Acute tubular necrosis and interstitial nephritis is present. H&E stain. B, Warthin-Starry
showing low numbers of leptospires in the renal interstitium (arrow). (Images courtesy Dr.
Patricia Pesavento, University of California, Davis Veterinary Anatomic Pathology Service.)
B
FIGURE 50-6  Histopathology of the liver in leptospirosis. A, Minimal hepatic changes
and severe cholestasis in an 8-year-old male neutered Australian shepherd mix that was
euthanized because of anuric leptospirosis. The activity of serum ALP and the serum total
titer to serovar Pomona (1:400 to >1:3200 12 days later). B, Liver plate disarray with hepa-
tocellular dissociation in a dog with leptospirosis. (B, Image courtesy John Cullen, North
Carolina State University and Jean-Jacques Fontaine, École Nationale Vétérinaire d’Alfort)

TABLE 50-5
Antimicrobials Recommended for Treatment of Leptospirosis
recommends that all dogs with leptospirosis should be treated in Dogs
with doxycycline, 5 mg/kg q12h PO or IV for 2 weeks.20 If
vomiting or other adverse reactions preclude the use of doxy- Interval Duration
cycline, parenteral ampicillin or penicillin G can be used (Table Drug Dose Route (hours) (days)
50-5). Dogs initially treated with a penicillin derivative should
be treated with doxycycline for 2 weeks after gastrointestinal Doxycycline 5 mg/kg PO, IV 12 14
signs resolve. Fluoroquinolones are not highly efficacious for Ampicillin 20 mg/kg* IV 6 Variable
clearance of leptospires from tissues.56 Other antimicrobials Penicillin 25,000- IV 12 Variable
that have been efficacious in clinical trials involving human 40,000 U/kg
patients with leptospirosis include ceftriaxone, cefotaxime,
and azithromycin.57,58 *Reduce dose in renal failure.
482 SECTION 2  Bacterial Diseases
Supportive Care
Dogs with severe disease require aggressive treatment with IV
crystalloids, potassium supplementation, antiemetics, antihy-
pertensives, antacids, blood products, and parenteral or enteral
nutrition, with attention to precautions to minimize zoonotic
transmission (see Public Health Aspects, later). Referral to a
24-hour care facility, ideally with access to dialysis, should be
considered for severely ill dogs because of the need for intensive
monitoring and close observation. Electrolyte and acid-base sta-
tus may require frequent monitoring.
Fluid therapy is critical and should be calculated based on
measurement of “outs and ins,” together with careful attention
to body weight, respiratory rate, lung sounds, blood pressure,
and if possible, central venous pressure. Dogs with polyuric
renal failure may require extremely high fluid rates. For dogs
with oliguric or anuric renal failure, high fluid rates can lead
to overhydration and respiratory failure. Urine output in dogs
with oliguria or anuria should be closely monitored through
use of a closed, indwelling urinary catheter and collection bag
system. Diuretics such as furosemide and mannitol may be indi-
cated. Failure of urine production after adequate hydration is
achieved is an indication for dialysis (see Renal Replacement
Therapy, next). Once recovery of kidney values has occurred,
fluids should be tapered slowly before they are discontinued to
ensure that proper hydration status is maintained. In some dogs,
possibly because of enteritis or pancreatitis, inappetence persists
for several days after kidney and liver values normalize. Contin-
ued nutritional support is required during this period.
Dogs with SPHS may require oxygen therapy and, rarely,
mechanical ventilation. The optimum treatment for dogs
with SPHS is unknown. Human patients with SPHS have had
improved outcome following cyclophosphamide therapy and
plasma exchange.59
Renal Replacement Therapy
Early hemodialysis results in improved survival and shorter hos-
pital stays in human patients with leptospirosis.60 Renal replace-
ment therapy is indicated for dogs with inadequate urine output
that develop volume overload, hyperkalemia, a BUN greater than
80 mg/dL, or signs of uremia that do not respond to medical man-
agement.20 Approximately 50% of 89 dogs with leptospirosis at
the University of California, Davis, VMTH in the past 10 years
received hemodialysis; the median number of hemodialysis treat-
ments required was 3 (range, 1 to 14). Recovery of adequate renal
function usually occurs within 2 to 4 weeks of starting dialysis.
Prognosis
When the treatment is initiated early and aggressively, the prog-
nosis for recovery from acute leptospirosis is excellent. Survival
rates are in excess of 50%,41 and in centers where hemodialysis is
available, survival rates can exceed 80%. Precise survival rates for
dogs with SPHS are unknown but may be lower. Successful treat-
ment is associated with normalization of the platelet count, serum
BUN and creatinine concentrations, and serum liver enzyme activ-
ities within 10 to 14 days. Icterus may be slow to resolve, and
urine-concentrating ability may not return for at least 4 weeks.
Some dogs have residual permanent renal damage after recovery.
Immunity and Vaccination
Recurrence of leptospirosis in dogs after recovery from natural
infection appears to be rare, but the duration of immunity after
natural infection is unknown. Immunity after vaccination with
inactivated vaccines is serogroup specific and possibly serovar
specific, although partial immunity to heterologous serogroups
was shown in one report.61 A live attenuated LPS mutant L.
interrogans serovar Manilae vaccine was shown to induce
strong protection in hamsters against challenge either with the
same or a serologically unrelated (Pomona) serovar.62 After
challenge with serovar Pomona, survival rates were 100% in
hamsters immunized with the attenuated live vaccine, com-
pared with only 40% for those immunized with an inactivated
serovar Manilae vaccine.
Current vaccines for dogs are inactivated bacterins. Bivalent
vaccines that contained only serovars Icterohaemorrhagiae and
Canicola were first introduced for prevention of leptospirosis
in dogs. In the United States, these have now been replaced
with four-serovar vaccines that contain serovars Icterohaem-
orrhagiae, Canicola, Grippotyphosa, and Pomona. In Europe,
serovar Icterohaemorrhagiae and Canicola vaccines are still
available, but vaccines that contain serovar Icterohaemor-
rhagiae, Canicola, and Grippotyphosa (Pfizer Animal Health)
and serovar Copenhageni (serogroup Icterohaemorrhagiae),
Portland-vere (serogroup Canicola), Bratislava (serogroup Aus-
tralis), and Dadas (serogroup Grippotyphosa) (MSD Animal
Health) have been introduced. Leptospiral vaccines effectively
prevent disease and reduce shedding after challenge with the
serovar included in the vaccine. The duration of immunity fol-
lowing vaccination is at least 12 months.32,63,64 Leptospirosis
has occurred in North American dogs immunized with bivalent
vaccines; however, disease is rare in dogs immunized with four-
serovar vaccines.13
In the past, vaccination for with Leptospira vaccines has
been associated with type I hypersensitivity reactions such as
anaphylaxis, especially in small-breed dogs. Anecdotal evidence
from industry and veterinary practitioners in North America
suggests that the prevalence of these reactions has considerably
reduced in recent years (to <1%) following efforts from industry
to remove constituents that have been associated with vaccine
reactivity.20
Immunization is recommended for dogs at risk of expo-
sure. In some regions, this may be dogs that contact wild-
life or farm animals, or dogs that swim, wade, or drink from
environmental water sources. In other regions, dogs in urban
or suburban backyards may be at risk if there is significant
exposure to wildlife or rodents in the immediate home envi-
ronment. Immunization may be optimally performed a few
months before the onset of the peak season for leptospirosis
in a given geographic region. There is no evidence that the
initial series needs to be readministered to dogs that are over-
due for an annual booster. For dogs that experience natural
infection, immunization could commence a year after recov-
ery, although the duration of immunity after natural infection
and the degree and duration of cross-protection to heterolo-
gous serovars that follows natural infection requires further
investigation.
Prevention
Leptospirosis can be prevented by restricting dogs’ access to
potential reservoir host species or environmental water sources.
Access to wildlife and farm animals can be reduced through
fencing and rodent control. Where this is not possible, immuni-
zation is recommended.

Public Health Aspects
In humans, leptospirosis occurs after an incubation period of 2
to 20 days (mean, 10 days). In 90% of infections, it is a mild,
influenza-like illness. Less commonly, it is manifested by severe
multiorgan failure, with renal failure, hepatic failure, or SPHS.
Abortion can occur during pregnancy. Weil’s disease, which
is characterized by impaired renal and hepatic function, usu-
ally occurs a week after an initial illness characterized by fever,
myalgia, headache, chills, and conjunctivitis.1
In developed countries, most human leptospirosis cases fol-
low recreational activities that involve water, such as house-
boating or participation in triathlons, caving, or white-water
rafting.65,66 Individuals who contact farm animals can also
develop leptospirosis. Contact with wild rodents adopted as pets
has also led to human disease.67 In developing countries, stray
dogs may be a source of human infection, although rodents are
also important in this situation.68 The extent to which domestic
cats might serve as reservoir hosts is not understood.
Reports of transmission from clinically affected incidental
hosts (as opposed to subclinically infected reservoir hosts) to
other animals are rare, and there are no reports that document
transmission of leptospires from pet dogs to humans with the
support of molecular typing methods. Appropriate antimicro-
bial therapy may also reduce the possibility of transmission to
humans. Nevertheless, precautions should be taken in the hospi-
tal environment to minimize zoonotic transmission (see Chapter
11). All dogs with acute renal failure should be handled as lep-
tospirosis suspects until an alternative diagnosis is established.
Suggested precautions are shown in Box 50-1. Urine from dogs
with leptospirosis can be inactivated with a variety of disin-
fectant solutions, including iodine-based disinfectants, quater-
nary ammonium solutions, and accelerated hydrogen peroxide.
Large volumes of urine can be inactivated by 1:1 dilution with
a 10% solution of household bleach. In dogs with indwelling
urinary catheters, 10% bleach should be injected directly into
the collection bag before disposal. All blood, urine, and tissues
from dogs should be treated as medical waste. If a dog dies or
is euthanized, individuals who are to handle the remains should
be alerted of the possibility of leptospirosis.
Owners of affected dogs should be educated in regard to the
zoonotic nature of the disease. The risk of infection to owners
is probably low, because urinary shedding generally does not
occur until 7 to 10 days after the onset of illness, and doxy-
cycline treatment likely results in clearance of bacteria from
the kidneys and urine within 2 to 3 days. Nevertheless, until
antimicrobial therapy is completed, owners should avoid con-
tact with their dog’s urine. Routine household disinfectants
CHAPTER 50  Leptospirosis 483
BOX 50-1
Suggested In-Hospital Precautions for Dogs with Known
or Suspected Leptospirosis
Warning signage on the cage or run
Minimize movement around the hospital, avoiding trans-
port along common hallways
Gloves and disposable gowns
Protective eyewear and face shields if aerosolization of
urine likely
Handwashing before and after handling
No pregnant or immunocompromised staff to work with
the patient
Prompt disinfection of urine and urine spills
Treatment with appropriate antimicrobial drugs
Indwelling rather than intermittent urinary catheterization
for monitoring urine output
Dogs walked or gurneyed outside to urinate frequently if
urinary catheter not in place
Urination outside in a designated area that can be easily
disinfected
Warnings to others who handle the dog or specimens from
the dog
and gloved hands should be used to clean up urinary accidents
that occur indoors. Dogs should be taken to urinate in a place
that is away from water and away from other pets and peo-
ple, especially children. Owners should wash their hands after
they handle their pets. Veterinarians should recommend that
owners seek medical attention if illness occurs around the time
their dog is diagnosed with leptospirosis, or if they have any
questions about the disease in humans. Immunocompromised
humans should be referred to their medical practitioners for
advice. Owners should also be informed that environmental
sources of infection may represent an ongoing risk, not only to
other dogs in the household but also to humans. If other dogs
in the household may have been exposed at the same time as the
dog diagnosed with leptospirosis, those dogs should be treated
with doxycycline for 2 weeks, because subclinical infection and
shedding may have occurred.20
Veterinarians should contact their local or state health
department or the Centers for Disease Control and Prevention
if additional questions arise regarding the public health risks
and zoonotic transmission of leptospirosis.20
484 SECTION 2  Bacterial Diseases
CASE EXAMPLE
FIGURE 50-7  “Makalu,” a 1.5-year-old male neutered kelpie dog that developed
leptospirosis.
Signalment: “Makalu,” a 1.5-year-old male neutered kelpie
dog from Dixon in northern California (Figure 50-7).
History: Makalu had a 1-day history of lethargy and inappetence.
The owner, a veterinarian, took his temperature at home and
it was >104°F (40°C). There had been no vomiting, diarrhea,
coughing, sneezing, or increased thirst and urination. Makalu
lived on a property with another dog, three cats, sheep, goats,
and a pot-bellied pig. He normally ate a commercial dry dog
food; he consumed a rawhide the day before he became
ill. There was no history of previous travel, toxins, trauma,
medications, or tick exposure. He was 3 months overdue for
vaccination but had been vaccinated as a puppy for distemper,
hepatitis, parvovirus, rabies, and leptospirosis. The owner did
not recall which Leptospira vaccines he received.
Physical Examination:
Body Weight: 22.4 kg
General: Quiet, alert and responsive. Ambulatory on all four limbs,
T = 103.7°F (39.8°C), HR = 80 beats/min, RR = 20 breaths/min,
mucous membranes pink, CRT = 1 s. Esti­mated to be 7% to 8%
dehydrated based on skin turgor.
Integument, Eyes, Ears, Nose, and Throat: Mild scleral
injection and chemosis were present bilaterally.
Musculoskeletal: Appeared stiff, but there was no evidence of
synovial effusion. Vocalized on flexion of the right thoracic
limb.
Cardiovascular: Strong femoral pulses. No murmurs or
arrhythmias auscultated. Systolic blood pressure was
145 mm Hg.
Respiratory: Quiet bronchovesicular sounds were present in
all lung fields.
Gastrointestinal and Genitourinary: A tense abdomen was
noted, with abdominal splinting noted on palpation. No
abnormalities were detected on rectal examination.
Lymph Nodes: No abnormalities were detected.
Laboratory Findings:
CBC:
HCT 41.4% (40%-55%)
MCV 70.2 fL (65-75 fL)
MCHC 36.0 g/dL (33-36 g/dL)
WBC 8820 cells/µL (6000-13,000 cells/µL)
Neutrophils 7673 cells/µL (3000-10,500 cells/µL)
Lymphocytes 353 cells/µL (1000-4000 cells/µL)
Monocytes 441 cells/µL (150-1200 cells/µL)
Platelets 74,000 platelets/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 143 mmol/L (145-154 mmol/L)
Potassium 3.7 mmol/L (4.1-5.3 mmol/L)
Chloride 104 mmol/L (105-116 mmol/L)
Bicarbonate 17 mmol/L (16-26 mmol/L)
Phosphorus 5.1 mg/dL (3.0-6.2 mg/dL)
Calcium 9.6 mg/dL (9.9-11.4 mg/dl)
BUN 90 mg/dL (8-31 mg/dL)
Creatinine 4.6 mg/dL (0.5-1.6 mg/dL)
Glucose 110 mg/dL (70-118 mg/dL)
Total protein 5.1 g/dL (5.4-7.4 g/dL)
Albumin 2.4 g/dL (2.9-4.2 g/dL)
Globulin 2.7 g/dL (2.3-4.4 g/dL)
ALT 42 U/L (19-67 U/L)
AST 146 U/L (21-54 U/L)
ALP 73 U/L (15-127 U/L)
Creatine kinase 1387 U/L (46-320 U/L)
GGT 2 U/L (0-6 U/L)
Cholesterol 189 mg/dL (135-345 mg/dL)
Total bilirubin 0.4 mg/dL (0-0.4 mg/dL).
Urinalysis: SGr 1.013; pH 7.5, 2+ protein (SSA), 1+ bilirubin, 3+
hemoprotein, 2+ glucose, 0 WBC/HPF, 20-30 RBC/HPF, rare
transitional epithelial cells.
Coagulation Profile: PT 8.8 (7.5-10.5), APTT 16.8 (9-12),
fibrinogen 280 (90-255).
Serum Ethylene Glycol Assay: Negative.
Imaging Findings:
Thoracic Radiographs: The cardiovascular and pulmonary
structures appeared within normal limits.
Abdominal Ultrasound: The liver and spleen were both
enlarged. The tail of the spleen had a mildly mottled
echotexture. Both kidneys were mildly enlarged, and
measured approximately 8 cm in length. The renal cortices
were mildly echogenic. A small amount of subcapsular and
retroperitoneal fluid was noted in association with both
kidneys. The mesenteric lymph nodes were prominent.
Microbiologic Testing: Aerobic bacterial culture of blood
and urine: No growth.
Leptospira serology (MAT): Day 2 after the onset of illness, nega-
tive for antibodies to serovars Canicola, Grippotyphosa,
Hardjo, Icterohaemorrhagiae, and Pomona at 1:100. Posi-
tive for antibodies to serovar Bratislava at 1:100. Day 10 af-
ter the onset of illness, negative for antibodies to serovar
Canicola. Positive for antibodies to serovars Grippotyphosa
(1:400), Hardjo (1:100), Icterohaemorrhagiae (1:800), Pomo-
na (1:12,800), and Bratislava (1:3200).
Diagnosis: Leptospirosis.
Treatment: Makalu was aggressively treated with intra­venous
fluids, mannitol, famotidine, a metoclopramide, and ampicillin
(20 mg/kg IV q6h). Despite this treatment and administration
of furosemide, urine output remained at <0.8 mL/kg/hr. After
18 hours of hospitalization, dialysis was initiated. After a second
dialysis treatment 2 days later, polyuria ensued (Table 50-6). On
day 3, DIC developed, with further prolongation of the PT (14.3
s) and APTT (21.1 s), hypofibrinogenemia (<90 mg/dL), and
a positive D-dimer assay. The dog was treated with multiple
 CHAPTER 50  Leptospirosis 485

units of fresh frozen plasma. Treatment with ondansetron was
also initiated on day 3 because of persistent vomiting, and
total parenteral nutrition was instituted on day 4. Makalu’s
mentation improved on day 5 and he began eating on day 8.
Comments: Antibody titers in this dog were very low or  nega­
tive initially because insufficient time had elapsed for
antibody production to occur. The diagnosis of leptospirosis
was based on consistent clinical signs together with
seroconversion on day 10. Liver enzyme activities were
not increased in this dog on admission, but increased
subsequently. Without dialysis, this dog would have died
from the disease.

TABLE 50-6
Serial Changes in Clinicopathologic Variables in a Hospitalized Dog with Leptospirosis That Was Treated with Hemodialysis
Variable Day 1 Day 3 Day 4 Day 5 Day 8 Day 10
ALT (U/L) 62 ND 105 110 82 71
ALP (U/L) 83 ND 97 97 79 75
Bilirubin (mg/dL) 0.4 ND 0.5 0.8 0.3 0.3
BUN (mg/dL) 105 144 54 47 35 27
(27)* (30)
Creatinine (mg/dL) 5.6 12.9 4.8 3.3 2.1 1.6
(2.2) (3.1)
Albumin (g/dL) 2.2 2.4 2.5 2.9 3.0 3.1
Platelets (x1000/µL) 74 ND 26 91 92 clumped
Urine output (mL/kg/hr) <0.8 0.25 10 10 7 ND

*Values in parentheses are postdialysis values. ND, not done.

SUGGESTED READINGS
Araujo ER, Seguro AC, Spichler A, et al. Acute kidney injury in human
leptospirosis: an immunohistochemical study with pathophysiologi-
cal correlation. Virchows Arch. 2010;456(4):367-375.
Sykes JE, Hartmann K, Lunn KF, et al. 2010 ACVIM small animal con-
sensus statement on leptospirosis: diagnosis, epidemiology, treatment
and prevention. J Vet Intern Med. 2011;25(1):1-13.
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CHAPTER 51
Lyme Borreliosis
Jane E. Sykes
Overview of Lyme Borreliosis
First Described: Connecticut, USA, 1982 (Burgdorfer)1
Cause: Borrelia burgdorferi sensu lato (a spirochete)
Primary Mode of Transmission: Ixodes spp. ticks
Affected Hosts: Humans and a large variety of animals; dis-
ease occurs in dogs, humans, horses, cattle
Geographic Distribution: North America, Europe, Asia
Major Clinical Signs: Fever, lethargy, inappetence, lameness
due to polyarthritis. Signs of Lyme nephropathy include
vomiting, weight loss, and polyuria and polydipsia.
Differential Diagnoses: Differential diagnoses for suspected
Lyme polyarthritis includes bilateral cruciate ligament rup-
ture, primary immune-mediated polyarthritis, septic poly-
arthritis, and polyarthritis secondary to infection with other
pathogens such as Ehrlichia ewingii, Bartonella spp., Ehrlichia
canis, Anaplasma phagocytophilum, Rickettsia rickettsii,
and fungal organisms. Differential diagnosis for suspected
Lyme nephritis include leptospirosis, bacterial pyelone-
phritis, primary immune-mediated glomerulonephritis,
familial nephropathies, amyloidosis, and glomerulonephri-
tis secondary to other chronic infections such as Dirofilaria
immitis, Babesia canis, E. canis, and Leishmania spp. infections.
Human Health Significance: Dogs and cats are not a direct
source of human infection but may bring unfed infected
ticks into the house. Evidence of canine exposure to
B. burgdorferi is a sentinel for human exposure.
Etiologic Agent and Epidemiology
Lyme borreliosis, or Lyme disease, is a vector-borne spirochet-
osis caused by the motile, corkscrew-shaped bacterium Borrelia
burgdorferi sensu lato. Lyme disease occurs in North America,
Europe, and Asia. It was named after Old Lyme in Connecticut,
where clusters of the disease were first recognized in children
with juvenile rheumatoid arthritis in 1976.2 However, chronic
cutaneous manifestations of infection were recognized in human
patients in Germany as far back as 1883.3 The spirochete was
first detected in ticks by Burgdorfer in 1982.1 A variety of differ-
ent mammalian hosts and humans may be infected. The disease
has been increasingly recognized and is now the most common
vector-borne infectious disease of humans in the United States
and Europe. Reforestation of farmland and proliferation of deer
and tick populations may have contributed to emergence of the
disease in humans.4 Cats can be infected and seroconvert, but
appear to be relatively resistant to development of disease.5
Dogs can develop fever, arthritis, and renal disease, but most
infected dogs show no signs of illness.
More than 15 different species of B. burgdorferi sensu lato
have been described, some of which are nonpathogenic (Table
51-1). Borrelia turicatae, the cause of tick-borne relapsing fever
in humans, has been detected in sick dogs from Texas.6 The
DNA of Borrelia lonestari, which is thought to be nonpatho-
genic, has been detected in dogs from Arkansas.7 In North
America, Lyme disease is caused by Borrelia burgdorferi sensu
stricto, whereas in Europe and Asia, other species that belong
to Borrelia burgdorferi sensu lato are more important, spe-
cifically Borrelia afzelii and Borrelia garinii. This may account
for differences in clinical manifestations that occur in human
patients in Europe compared with those in North America (see
Public Health Significance, later). Some strains of B. burgdorferi
appear to have increased virulence, such as B. burgdorferi OspC
type A within the United States.8
B. burgdorferi sensu lato is transmitted by Ixodes ricinus-
persulcatus complex ticks (Table 51-2). The geographic distri-
bution of Lyme disease reflects that of the vector ticks as well
as the competency of the reservoir hosts involved. In the United
States, the vectors are Ixodes scapularis in the east and upper
Midwest, and Ixodes pacificus in the West (see Chapter 29).
Major foci of infection exist in the upper Midwest, Northeast,
mid-Atlantic, and parts of northern California (Figure 51-1).9
In some endemic areas, seroprevalence in dogs is nearly 90%,10
although seroprevalence in a study that used a C6 ELISA assay
(see Diagnosis, later) was lower than this in endemic areas such
as Connecticut and Massachusetts, around 20%.11 Infection is
passed transstadially within the tick (i.e., from larva to nymph
to adult), and not transovarially (from adult to egg). Reser-
voirs for the spirochete in the Northeast and the upper Mid-
west are Peromyscus leucopus, the white-footed mouse, which
can harbor large numbers of the organism without overt signs
of illness,12 as well as shrews and chipmunks.13 In the western
United States, the western gray squirrel is the primary reser-
voir host.14 Lyme disease is less prevalent in the western United
States because I. pacificus prefers to feed on the western fence
lizard, a poor reservoir for the spirochete. Similarly, the low
prevalence of Lyme disease in the southeastern United States,
is because I. scapularis ticks feed primarily on lizards in this
region.15 B. burgdorferi is primarily transmitted to humans
by nymphal ticks, because they are extremely small and often
enough go unnoticed. Adult ticks may be more important for
transmission of infection to dogs.16 The nymphs of I. scapularis
quest in the late spring and summer, when humans and dogs are
often outdoors and become exposed. The peak questing times
for I. scapularis adult ticks are in the spring and fall. Other
487
488 SECTION 2  Bacterial Diseases

TABLE 51-1
Pathogenic and Nonpathogenic Species That Belong to Borrelia
burgdorferi sensu lato and Their Geographic Distributions
United States Europe Asia
Pathogenic B. burgdorferi B. burgdorferi B. afzelii
sensu stricto sensu stricto B. garinii
B. afzelii
B. garinii
Non-pathogenic B. bissettii B. valaisiana* B. bissettii*
or question- B. andersonii B. bissettii* B. japonica
able patho­ B. californiensis B. spielmanii* B. turdi FIGURE 51-1  Approximate geographic distribution of Lyme borreliosis in humans
genicity B. carolinensis B. lusitaniae B. tanukii (and dogs) in the United States.
B. americana B. bavariensis B. sinica
B. kurtenbachii B. yangtze

*B. valaisiana and B. bissettii have been isolated from single cases of
Lyme borreliosis; B. spielmanii has been detected in early skin lesions
(Stanek G, Reiter M. The expanding Lyme Borrelia complex—clinical
significance of genomic species? Clin Microbiol Infect 2011;17:487-493).

TABLE 51-2
Vectors of Lyme Disease Worldwide
Geographic Tick Common Reservoir host
Location Species Name for Borrelia
Western United Ixodes Pacific black- Western gray
States ­pacificus legged tick squirrel
Northeastern Ixodes Black-legged White-footed
and upper scapularis tick mouse, shrews,
­Midwestern ­chipmunks
United States
Europe Ixodes Castor bean Squirrels,
ricinus or sheep thrushes,
tick chipmunks,
mice, shrews,
hedgehogs,
rats, hares,
pheasants, FIGURE 51-2  Countries and regions where Lyme borreliosis occurs in Europe. The
voles size of the red dot correlates with the relative prevalence of the infection in those areas.
Asia Ixodes Taiga tick Voles
persulcatus region. A huge variety of reservoir hosts appear to be involved in
Europe. Small rodents such as squirrels appear to be an important
reservoir host for B. afzelii, and birds such as blackbirds and song
vector-borne pathogens, such as Anaplasma phagocytophilum, thrushes are important reservoir hosts for B. garinii.21
may be co-transmitted and complicate the clinical picture. There The complete genome of B. burgdorferi has been sequenced.
is some evidence that co-infection with A. phagocytophilum can The organism expresses different outer-surface lipoproteins at
enhance the pathogenicity of B. burgdorferi.17 different stages of infection, which allows it to adapt to dra-
In continental Europe, Lyme disease is most prevalent in cen- matically different environments within the arthropod vector
tral and eastern Europe, especially Poland, Slovakia, and Slovenia, and the mammalian host. This property of the organism has been
but also Germany, Switzerland, Austria, and southern Scandina- exploited for the purpose of diagnostic assay and vaccine devel-
via (Figure 51-2). B. garinii was detected in a dog from the Czech opment. Outer surface proteins of importance include OspA,
republic with meningoencephalitis,18 and B. afzelii has been OspC, and VlsE. Over the fall, winter, and spring, the spirochete
detected in dogs from Poland.19 Evidence of infection has also remains dormant within the nymphal tick and expresses OspA,
been found in dogs from the British Isles,20 where endemic areas which allows it to adhere to the tick midgut. When the tick ingests
include the Scottish Highlands, Ireland, Wales, the Lake District, mammalian blood in the late spring and summer, OspA expres-
the Yorkshire Moors, Exmoor, Wiltshire, Berkshire, and particu- sion is downregulated by the spirochete, and OspC expression is
larly the New Forest, the South Downs, and the Thetford Forest upregulated.22 The spirochete then moves to the tick hemolymph

and salivary glands. OspC binds a tick salivary gland protein,
which may help the organism evade the host immune response.
OspC also binds to mammalian plasminogen and helps the spiro-
chete to disseminate within the mammalian host.23 VlsE under-
goes recombinational shuffling of its genetic code, which further
allows the spirochete to evade the immune response.24 Finally,
B. burgdorferi may undergo metamorphosis into a spherical
shape when it encounters unfavorable conditions within the host,
such as when it is exposed to antibiotics, nutrient deprivation,
and changes in pH.25 This may also contribute to its ability to
evade the immune system and resist antimicrobial drug treatment.
Clinical Features
Signs and Their Pathogenesis
Transmission is thought to require tick attachment for a mini-
mum of 24 hours,26 but in some cases transmission may occur
at earlier time points.27 The spirochete replicates at the site
of tick attachment, then disseminates to multiple locations.
Although it can be transiently found in blood, the organism pri-
marily replicates and spreads through connective tissue (Figure
51-3). It binds proteins such as plasminogen, β3 integrins such
as the platelet integrin αIIbβ3, glycosaminoglycans, fibronectin,
laminin, and decorin (a collagen proteoglycan).28 After inva-
sion, the organism can persist in dogs for over a year, through
evasion of host immune responses.29
In contrast to human Lyme disease, dogs do not develop an early
skin rash (erythema migrans). It has been estimated that only 10%
of affected dogs show overt signs of illness. Initial signs in dogs
occur 2 to 5 months after a tick bite and consist of variable fever,
inappetence, thrombocytopenia, and lameness due to neutrophilic
polyarthritis. In a study of experimentally infected dogs, the first
joint to be affected was the joint closest to the site of the tick bite,
which supports the notion that spirochetes reach the joints as a
result of spread through connective tissue.29,30 Subsequently, other
joints can be affected, and dogs may develop shifting lameness. It
is not clear whether dogs develop other clinical manifestations seen
in humans such as persistent, antibiotic-refractory Lyme arthritis;
uveitis; carditis; or encephalopathy. Complete heart block was
described in a seropositive dog from Connecticut that had patho-
logic changes at necropsy consistent with Lyme carditis.31
Lyme nephritis is a syndrome of membranoproliferative glo-
merulonephritis that has been recognized in dogs in association
with exposure to B. burgdorferi. Rare reports of a similar dis-
ease in human patients exist.32 Golden and Labrador retriever
breeds appear to be overrepresented,33 and it has been suggested
that Shetland sheepdogs and Bernese mountain dogs might also
be predisposed.34-36 Dogs with Lyme nephritis are younger than
dogs with other glomerular diseases. In one study, more than 50%
of dogs with Lyme nephritis were 5 years of age or younger.33
Many affected dogs are also thrombocytopenic, and some also
have polyarthritis.33 The pathogenesis of Lyme nephritis is uncer-
tain; immune-mediated mechanisms have been proposed.37 The
DNA or antigen of the spirochete cannot be consistently detected
within renal biopsies.37,38 Subendothelial deposits of IgM, IgG,
and C3 can be detected within the glomeruli.33 Clinical signs
result from proteinuria and renal failure and include inappetence,
lethargy, weight loss, vomiting, and polyuria and polydipsia.
Physical Examination Findings
Physical examination findings in dogs with Lyme arthritis include
lethargy, fever, lameness, and swollen, warm, painful joints. Mild
generalized peripheral lymphadenopathy may be present. Dogs
CHAPTER 51  Lyme Borreliosis 489
FIGURE 51-3  Borrelia burgdorferi in the metatarsal tendon sheath of a mouse with
severe combined immunodeficiency. (Courtesy Dr. Stephen Barthold, Center for Compara-
tive Medicine, University of California, Davis.)
with Lyme nephritis may have a thin body condition, dehydration,
and show evidence of peripheral edema, pleural effusion, or asci-
tes, especially after crystalloid fluid administration. Complications
of hypertension can sometimes occur, such as retinal detachment.
Diagnosis
Diagnosis of Lyme disease is notoriously difficult. The over-
whelming majority of infected dogs experience subclinical infec-
tion, after which positive antibody titers may persist for months
or years. Because only a small percentage (e.g., <10%) of infec-
tions result in disease, it is important to distinguish between
diagnosis of infection or previous exposure to the spirochete and
diagnosis of Lyme disease. Dogs with positive antibody titers may
develop unrelated illnesses, which results in diagnostic confusion.
As in human patients, the diagnosis must be established based on
characteristic clinical signs, a history of exposure in an endemic
area, and a positive antibody response to B. burgdorferi.39 A his-
tory of tick exposure may not always be present.
Laboratory Abnormalities
Complete Blood Count
The most common finding on the CBC of dogs with Lyme poly-
arthritis or nephritis is mild to moderate thrombocytopenia.
The mechanism of thrombocytopenia is not known, but it may
be immune mediated, because treatment of dogs with Lyme
nephritis with immunosuppressive drugs can result in normal-
ization of the platelet count. Mild to moderate anemia and leu-
kocytosis due to a mature neutrophilia may be present in dogs
with Lyme nephritis, but white cell counts may also be normal.
Lymphopenia and mild neutropenia can also occur.
Serum Biochemical Tests
Dogs with polyarthritis typically have minimal changes on serum
biochemistry testing. Dogs with Lyme nephritis may show azo-
temia, mild to marked hypoalbuminemia, metabolic acidosis,
and electrolyte changes such as hyperphosphatemia, hypochlo-
remia, and mild hyperkalemia or hypokalemia. Uncommonly,
hypercholesterolemia is present.
Urinalysis
The urinalysis in dogs with Lyme nephritis generally reveals
isosthenuria and proteinuria. Pyuria and microscopic hematuria
490 SECTION 2  Bacterial Diseases

may also be present. Urine protein to creatinine ratios are often
greater than 5 and may be above 15 in some dogs.
Coagulation Profile
Dogs with Lyme nephritis may have low antithrombin activities
as a result of glomerular antithrombin loss, which may lead to
hypercoagulability syndromes. Sometimes, abnormalities such
as a shortened PT, prolonged APTT, and increased fibrinogen
and D-dimer concentrations are present.40
Synovial Fluid Cytology
Dogs with Lyme arthritis may have normal synovial fluid cytol-
ogy,30 or there may be markedly increased numbers of nonde-
generate neutrophils within the synovial fluid of distal joints
(>5000 and frequently >10,000 cells/µL). Because not all joints
may be affected, synovial fluid should be collected from at least
three and preferably four peripheral joints.
Diagnostic Imaging
Plain Radiography
Lyme arthritis is a nonerosive polyarthritis, so the only changes
visible on plain radiography of the joints are increased periar-
ticular soft tissue opacity. Thoracic radiography is generally
unremarkable, but pleural effusion is occasionally identified in
severely hypoalbuminemic dogs with Lyme nephritis.
Sonographic Findings
Abdominal sonographic examination is normal in dogs with
Lyme arthritis. In dogs with nephritis, thickening and increased
echogenicity of the renal cortices, decreased renal corticomedul-
lary distinction, and peritoneal effusion may be seen.
Microbiologic Tests
Specific diagnostic assays available for Lyme borreliosis in dogs
are described in Table 51-3.

TABLE 51-3
Diagnostic Assays Available for Lyme Borreliosis in Dogs
Assay Specimen Type Target Performance
Bacterial isolation Skin biopsy close to B. burgdorferi spirochete Low sensitivity, requires special media, and may take sev-
the tick bite site; eral weeks. Isolation does not imply that B. burgdorferi
synovial fluid is the cause of disease.
Serology (C6 assay) Serum Antibodies against Positive serology does not equate with Lyme disease,
B. burgdorferi C6 so test results must be interpreted in light of clinical
protein findings. False positives can occur in regions of low
prevalence. False negatives are rare, because antibodies
are present by the time dogs develop illness. Cross-­
reactivity with vaccine antibodies does not occur.
Serology (whole cell Serum Antibodies against As for C6 serology except that cross-reactivity with vac-
IFA and ELISA) B. burgdorferi antigens cine antibodies occurs, and false positives may also
occur with other inflammatory diseases, and other
spirochete infections.
Serology (Western Serum Antibodies against Used to confirm the serological response to natural infec-
immunoblot) B. burgdorferi antigens tion in dogs that test positive with whole-cell ELISA
and IFA. Technically difficult to perform and requires
expertise to interpret. Positive serology does not equate
with Lyme disease, so test results must be interpreted in
light of clinical findings.
Serology (multiplex Serum Antibodies against Antibodies to OspC appear only early in infection, where-
fluorescent bead B. burgdorferi OspA, as those to OspF reflect chronic infection and appear
assay) OspC, and OspF to correlate with the C6 antibody response. Sensitivity
and specificity in dogs with naturally occurring Lyme
disease unknown. Positive serology does not equate
with Lyme disease, so test results must be interpreted in
light of clinical findings.
Serology (multi- Serum Antibodies against Sensitivity and specificity in dogs with naturally occurring
target silicon disc– B. burgdorferi OspA, Lyme disease requires further study. Positive serology
based assay) OspC, and OspF, SLP, does not equate with Lyme disease, so test results must
and P39 be interpreted in light of clinical findings.
PCR As for isolation B. burgdorferi DNA Rapid but insensitive. Synovial fluid from dogs with
polyarthritis may be the best specimen, but further
study is needed. Assay performance can vary between
laboratories.
 CHAPTER 51  Lyme Borreliosis 491

Culture A multiplex fluorescent bead assay has been marketed in

B. burgdorferi can be isolated from tissue specimens in Barbour-
Stoenner-Kelly medium. The optimal specimen for culture is a
skin biopsy collected from a site adjacent to the tick bite, which
is rarely identifiable in affected dogs. Incubation of cultures for
several weeks may be required. Because of these factors, culture
is not generally used on a routine basis for diagnosis.
Serologic Diagnosis
Serologic tests for antibody are the main assays used for diagno-
sis of Lyme disease in humans and also in dogs, but it is critical
to recognize that positive serology does not necessarily equate
with the presence of Lyme disease. In Europe, infection with
North America for detection of antibodies to three antigens
of B. burgdorferi: OspA, OspC, and OspF.43 The assay uses
tiny beads to which OspA, OspC, and OspF are coupled. Dog
serum is added to the beads, and if present, antibodies in the
serum bind to the antigens and can be detected using a fluores-
cent conjugate. The pattern of reactivity to each antigen can be
used to differentiate among the response to vaccination, early
infection, and chronic infection. The presence of anti-OspA
antibodies suggests previous vaccination, because vaccines
contain OspA, and the spirochete rarely expresses OspA within

Vaccination  infection
nonpathogenic variants of B. burgdorferi sensu lato can result in

Vaccination rOspA
positive serologic results, which further complicates diagnosis.

Vaccination lysate
Because the infection is chronic and persistent, and the incuba-

Control OspA
tion period is long, paired serology is generally not performed,
because seroconversion may not occur. However, paired serol-

Negative

Infection
Control
ogy using serologic panels that include B. burgdorferi may be
required for diagnosis of other vector-borne diseases that might
be present. Positive test results in regions of low prevalence are
more likely to be false positives than in regions of high preva-
lence, so interpretation of positive results should be performed
with care in low-prevalence regions.
Markline
Currently, one of the most widely used serodiagnostic tests for Serum control
canine Lyme disease is based on a C6 ELISA, which detects anti- lgG
bodies against a portion of the VlsE lipoprotein. The advantages Conjugate
lgA
control
of the C6 ELISA assay are that (1) it detects IgG antibodies 3 to lgM
5 weeks after the time of infection, so by the time dogs develop VlsE
clinical signs they are virtually always seropositive,41 and (2) it
is negative in dogs that have been vaccinated for Lyme disease,
because the antigen is not expressed by organisms used in Lyme P83/100
vaccines. The C6 ELISA is available as an in-practice lateral-flow
assay, in combination with serodiagnostic spots for Ehrlichia
canis/Ehrlichia ewingii antibody, Anaplasma spp. antibody, and
p58
Dirofilaria immitis antigen (SNAP 4Dx Plus, IDEXX Labora-
tories, ME) and as a quantitative ELISA (Quant C6), which is
performed at IDEXX central veterinary diagnostic laboratories.
p43
There is no correlation between the magnitude of the C6 ELISA
titer and disease severity. In a study from Europe, positive test FlaB p41
results did not correlate with disease.36 Another rapid immuno- BmpA p39
chromatographic ELISA assay has recently become available in
the United States (Abaxis VetScan Canine Lyme Rapid Test) that
OspA
detects antibodies to a different portion of the VlsE lipoprotein,
OspC and p41; these are combined on a single line. p30
Other serologic assays that are available include whole-
cell ELISA or immunofluorescent antibody (IFA) assays and OspC
Western blotting (WB; see Chapter 2 for a description of these
techniques). WB has traditionally been considered the gold
standard assay. False positives using whole-cell ELISA and p21
IFA have the potential to occur in patients with other spiro-
chete infections, with other inflammatory disorders, and in Osp17
vaccinated dogs. In human patients, a two-tiered approach is
recommended for diagnosis.39 Serum from patients who test
positive for IgM or IgG with ELISA is then subjected to WB,
because WB has increased specificity. WB has been used to dif- FIGURE 51-4  Western immunoblots of sera from dogs not infected with B. burgdor-
feri (Negative); immunized with recombinant OspA (vaccination rOspA); Immunized with a
ferentiate the response to immunization and natural infection
whole cell lysate vaccine (vaccination lysate); immunized and infected with B. burgdorferi
in dogs (Figure 51-4).42 It has also been used to identify “dual (vaccination + infection); and infected only with B. burgdorferi (infection). The left strip
status” dogs, that is, dogs that have been both immunized and shows all major signals available on the blot (control); the second strip from the left is
naturally infected. WB is more time-consuming to perform stained with a monoclonal antibody against OspA (control OspA). (Courtesy R. Straubinger,
than ELISA and IFA and experience is required to interpret it Ludwig-Maximillians-Universitat, Munich, Germany. In: Greene CE: Infection Diseases of
correctly.43 the Dog and Cat. 4th ed. St. Louis: Elsevier/Saunders; 2012.)
492 SECTION 2  Bacterial Diseases
the host. OspC is expressed as the spirochete moves to the tick
salivary glands and shortly after it enters the host. Thus an
antibody response to OspC may suggest recent infection; titers
decline and become undetectable beyond 3 months after infec-
tion.44 Antibodies to OspC appear as early as 3 weeks after
experimental infection of dogs, and OspF as early as 5 weeks.44
OspF is expressed in more chronic infections, and can be
detected together with the C6 antibody response in naturally
exposed dogs (Figure 51-5).44,45 In dogs, when WB was used
as the gold standard, the sensitivities of the OspA, OspC, and
OspF assays were 83%, 62%, and 82%, and the specificities
were 90%, 89%, and 86%, respectively. The use of WB as
the gold standard was questioned, and it was suggested that
in fact, the fluorescent bead assay may have greater sensitivity
and specificity than WB. The performance of the fluorescent
bead assay is yet to be thoroughly evaluated in dogs with natu-
rally occurring Lyme disease.
A novel silicon disc–based serologic assay is available in
the United States for detection of antibodies to B. burgdorferi,
E. canis, and A. phagocytophilum and antigen to Dirofilaria
immitis (Accuplex 4, Antech Diagnostics, Irvine, CA). The assay
for B. burgdorferi detects antibodies to the spirochete proteins
OspA, OspC, OspF, P39, and SLP and is performed at central
veterinary diagnostic laboratories. Preliminary data suggests
that laboratory analysis of the response to these proteins cor-
relates with the results of WB and can differentiate between the
responses to natural infection and vaccination, and acute and
chronic infection.46
Molecular Diagnosis Using the Polymerase Chain Reaction
As with culture, PCR assays are best performed on skin biopsy
specimens collected from a region adjacent to the tick bite site.
In human patients, the use of PCR assays for diagnosis of Lyme
borreliosis on blood has low sensitivity, and so false negatives are
common when B. burgdorferi PCR assay is used as part of a whole-
blood vector-borne infection PCR panel. Synovial fluid or synovial
membrane biopsies may be the optimum specimen for diagnosis
of Lyme arthritis in dogs. This is also true in human patients.47
However, in a study of experimentally infected dogs, PCR assay of
the synovial fluid was insensitive.30 The sensitivity and specificity
of PCR assays when used on specimens such as synovial fluid for
diagnosis of canine Lyme borreliosis requires further study.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with Lyme arthritis include
peripheral lymphadenomegaly, joint swelling, and synovial
effusion.29 The synovial fluid may be yellow-tinged and cloudy,
with decreased viscosity. In dogs with nephritis, the kidneys
are diffusely light tan and may have pinpoint red foci over the
cortical surfaces.33 In addition, evidence of systemic edema,
thrombosis and infarction, and uremia (such as parathyroid
hyperplasia, ulcerative stomatitis, and gastritis) may be present.
Histopathologic Findings
Histopathology of the joints of dogs that have been experimen-
tally infected with B. burgdorferi reveals fibrinosuppurative or
lymphoplasmacytic inflammation of the synovial membranes,
joint capsules, and tendon sheaths.29,30 Inflammation can some-
times be found in the joints even when a history of lameness
is not present.29 In human patients, organisms have been dem-
onstrated in the synovium using immunostains and electron
Antibody titer
C6
OspF
OspC
Infection 3mon
Time
FIGURE 51-5  Proposed canine antibody response to OspC, OspF, and C6 antigens
of B. burgdorferi during early and late infection. Data were obtained by fluorescent bead
multiplex analysis. The lines for the first 3 months after infection are based on multiplex
results from experimentally infected dogs. After 3 months the lines are projected from
data obtained from patient sera. The horizontal dotted line shows the cutoff value for the
multiplex assay. The vertical dotted line indicates 3 months after infection. (Modified from
Wagner B, Freer H, Rollins A, et al. Antibodies to Borrelia burgdorferi OspA, OspC, OspF,
and C6 antigens as markers for early and late infection in dogs. Clin Vaccine Immunol
2012;19:527-535.)
microscopy.48 Other findings in experimentally infected dogs
include periarteritis and perineuritis, especially within joint
capsules and the skin, as well as lymphoid hyperplasia within
peripheral lymph nodes. Mild, focal meningitis and encephalitis
was identified in dogs that were infected with B. burgdorferi
and immunosuppressed with dexamethasone.49
Renal histopathology in dogs with Lyme nephritis reveals dif-
fuse membranoproliferative glomerulonephritis (Figure 51-6),
dilation of the cortical renal tubules, tubular necrosis and regen-
eration, and mild to moderate, diffuse interstitial lymphoplas-
macytic inflammation.33 Periglomerular or diffuse interstitial
fibrosis may be present in dogs with end-stage disease.
Treatment and Prognosis
Antimicrobial Treatment
Antibiotic treatment is recommended for seropositive dogs that
have clinical illness consistent with Lyme disease. There is no
evidence that treatment of healthy seropositive dogs is beneficial
and it may lead to drug adverse effects and contribute to antimi-
crobial resistance in other bacteria and antimicrobial shortages.
The antibiotic of choice for Lyme arthritis is doxycycline. The
optimal dose and duration of treatment is unknown. Recom-
mended doses have included 5 mg/kg PO q12h and 10 mg/kg
PO q12h or 10 mg/kg PO q24h.34,50 Four weeks of treatment
has been recommended34 because the clinical manifestations of
disease resemble those of late-stage disease in human patients,
for which relapses occur when treatment durations of less than
30 days are used.39 Dogs with polyarthritis generally respond
clinically to doxycycline treatment within 24 to 48 hours. Other
differential diagnoses for polyarthritis should be considered if
an inadequate response to treatment occurs. On the other hand,
a clinical response to doxycycline treatment is not sufficient to
make a diagnosis of Lyme arthritis. This is because there are
other doxycycline-responsive causes of infectious polyarthritis
in dogs; signs of primary immune-mediated polyarthritis can
wax and wane; and doxycycline has antiinflammatory proper-
ties that may contribute to clinical improvement. For dogs that
do not tolerate doxycycline, amoxicillin can be used, which also
has activity against B. burgdorferi (Table 51-4). Azithromycin
and third-generation cephalosporins have also been used to
treat Lyme disease in human patients.39 Doxycycline treatment
 CHAPTER 51  Lyme Borreliosis 493

TABLE 51-4
Suitable Antimicrobials for Treatment of Lyme Disease in Dogs
Interval Duration
Drug Dose Route (hours) (days)
Doxycycline 5 to 10 mg/kg PO 12 28
Amoxicillin 20 mg/kg PO 8 28

TABLE 51-5
Immunosuppressive Drug Protocols That Could Be Considered
for Treatment of Lyme Nephritis in Dogs in Conjunction with
Antimicrobial Drug Treatment
FIGURE 51-6  Membranoproliferative glomerulonephritis in a 6-year-old, intact
male golden retriever dog that was seropositive for B. burgdorferi C6 protein antibodies Interval and
and had clinical and biochemical evidence of protein-losing nephropathy. The morpho- Drug Dose Route Duration
logic diagnosis was moderate to severe, diffuse, global membranoproliferative glomeru- Methylprednisolone 5 mg/kg IV 24 hours for
lonephritis with moderate chronic-active tubulointerstitial nephritis. The capillary walls sodium ­succinate 2 days
are thickened and there is mesangial cell proliferation. There was also evidence of focal with either
arteriolar mural disorganization, lamination, and sclerosis in this biopsy specimen. (Image
cyclophosphamide,
courtesy Dr. George Lees, Texas A&M University.)
mycophenolate
mofetil, or aza-
is also recommended for dogs with Lyme nephritis, although thioprine below
clinical improvement generally does not occur with antimicro- Cyclophosphamide 200 mg/m2 IV Every 14 days for
bial treatment alone (see Supportive Care). a maximum
Although treatment leads to clinical improvement and a reduc- of 6 cycles.
tion in antibody titers, B. burgdorferi can persist in tissues after Recheck CBC
treatment is discontinued. Studies in mouse and primate models 1 week after
have shown that despite negative cultures after treatment, PCR each treatment.
assays remain positive, and infection can be transmitted from
Mycophenolate 10 mg/kg IV or 12 hours until
PCR-positive to naïve animals.51,52 Regardless of the serologic
mofetil PO ­remission occurs,
test used, antibody titers can persist for months or years after res-
then consider
olution of clinical signs and antimicrobial drug treatment, so the
tapering
results of antibody testing are not consistently useful to guide fur-
ther treatment when clinical resolution of arthritis has occurred. Azathioprine 1-2 mg/kg PO 24 hours for 7
days, then every
Supportive Care 48 hours until
Dogs with arthritis usually respond rapidly to treatment with anti- remission occurs,
biotics, and additional treatment may not be necessary. Severe pain then consider
associated with arthritis may be treated with nonsteroidal antiin- tapering
flammatory drugs or opiate analgesics such as tramadol. Because
Lyme arthritis can be reactivated in some dogs by administration
of glucocorticoids more than a year after exposure,53 glucocor-
ticoids are not recommended for their antiinflammatory effects. Prognosis
Dogs with Lyme nephritis require management for protein- It is estimated that more than 90% of dogs that are infected
losing nephropathy, which may include treatment with intrave- with B. burgdorferi show no signs of illness. Dogs with Lyme
nous crystalloids and colloids, angiotensin-converting enzyme arthritis generally recover rapidly with antimicrobial treatment
inhibitors such as enalapril or benazepril, low-dose aspirin in an and do not develop relapse of disease. It is not clear whether
attempt to reduce thrombotic events, and nutritional support the “antibiotic-refractory arthritis” that occurs in a small per-
with a reduced protein diet. Placement of an esophagostomy or centage of genetically predisposed human patients also occurs in
gastrostomy tube for feeding purposes may be required. Blood dogs. Seropositive dogs with antibiotic-refractory polyarthritis
pressure should be monitored and if necessary, antihypertensive may have other unrelated causes of their disease, such as pri-
drugs such as amlodipine should be administered and titrated to mary immune-mediated polyarthritis.
effect. Anecdotally, improved outcomes have been noted in dogs The prognosis for dogs with Lyme nephritis is guarded to
with Lyme nephritis after immunosuppressive drug treatment poor. In the past, it was noted that most dogs die or are eutha-
(Table 51-5). The optimum protocol is yet to be determined. nized within days to weeks.34 Death often results from systemic
Dogs treated with immunosuppressive drugs should be carefully thrombosis or oliguric or anuric renal failure. Anecdotally, lon-
monitored for adverse effects of drug therapy. ger survival times of months to over a year have been noted in
494 SECTION 2  Bacterial Diseases
some dogs treated with immunosuppressive drugs in addition to
doxycycline and other supportive treatments.
Immunity and Vaccination
Borrelia spp. can evade the host immune response through mod-
ification of outer surface proteins and possibly metamorphosis
to a resistant spherical form.54 Interference with B-cell responses
by the spirochete also seems to occur.55 Humoral immunity
is most critical for resolution of infection, although T-cell
responses may be important for resolution of cell-mediated con-
sequences of infection, such as carditis in human patients.56
Several vaccines are available in North America for reduction
of Lyme disease in dogs. Lyme vaccines stimulate the formation of
borreliacidal antibodies that are directed against the surface pro-
teins normally expressed by the spirochete when it resides within
the tick. When the tick ingests dog blood that contains these anti-
bodies, complement-mediated lysis of the bacteria occurs within
the tick. Thus, bacteria are inactivated before they invade the host.
A canine recombinant OspA vaccine is available that induces the
formation of antibodies only against OspA. This subunit vaccine
is similar to a Lyme disease vaccine that was previously available
for prevention of human Lyme borreliosis (LYMErix, Glaxo-
SmithKline). In humans, the vaccine was shown to be safe and
efficacious, conferring immunity on 76% of adults and 100%
of children with a low prevalence of local injection site reactions
and flu-like symptoms.57 However, this vaccine was withdrawn
in February 2002 because of concerns that it may trigger autoim-
mune disease as a result of molecular mimicry in sensitive individ-
uals, despite a lack of scientific justification for these concerns.58,59
Two inactivated whole spirochete vaccines are also avail-
able for dogs. One contains two strains of B. burgdorferi, one
of which expresses high levels of OspC. The other vaccine is
a single-strain vaccine. The potential advantage of these vac-
cines is that they stimulate immunity not only to OspA, but
also to other surface proteins expressed by the spirochete. This
may provide the opportunity to neutralize B. burgdorferi when
OspA is downregulated, such as when the organism is in the
salivary glands and after it enters the host. Concern has been
expressed that whole-cell vaccines may be more likely to trig-
ger autoimmune consequences in dogs than the subunit vaccine,
especially in dogs that have been previously exposed to B. burg-
dorferi.34 However, no strong evidence exists that these types
of adverse reactions occur. Both OspA and whole-cell vaccines
provide protection against infection and disease that results
from experimental challenge.60,61 A study in an endemic area
for Lyme disease found a reduced prevalence of C6 seropositiv-
ity in dogs that had been vaccinated with a whole-cell bacterin
when compared with dogs that had not been vaccinated, sug-
gesting the possibility of protection from natural infection.62
The dual-strain bacterin has been shown to have a 1-year dura-
tion of immunity.63 Although no Lyme vaccine can be relied
on to provide complete protection, side-by-side comparisons of
canine Lyme vaccines that are available on the market have not
been performed. In addition, whether Lyme vaccination protects
against, or contributes to, the most severe consequence of infec-
tion, Lyme nephritis, is unknown. Finally, whether there are
any advantages or disadvantages of vaccinating dogs that are
already seropositive as a result of natural infection remains to be
elucidated. The recent identification of reinfection (as opposed
to relapse) as a cause of recurrent clinical signs of borreliosis
in human patients64 suggests that vaccination of previously
exposed individuals may offer some benefit, because the protec-
tion induced by vaccination (anti-OspA antibodies) differs from
that induced by natural infection (no anti-OspA antibodies).
However, whether ­recurrent Lyme disease occurs in dogs as a
result of reinfection is not known.
Prevention
Avoidance of tick-infested areas, use of topical ectoparasiti-
cides, and routine inspection of dogs for ticks after outdoor
activities can help to prevent Lyme disease. Ticks should be
removed within 24 hours of attachment, before transmission
of the spirochete can occur, but removal up to 60 hours after
attachment may still reduce the chance of transmission.65 Refer
to Chapter 28 for general information on tick prevention and
removal. Although treatment with a single dose of doxycycline
after a known Ixodes tick bite can prevent Lyme disease in
humans66 and mice,67 it is controversial because of the very low
risk of infection after a tick bite.68 A study in mice showed that
treatment 2 or more days after tick removal was ineffective.67
For healthy dogs that test positive for antibodies to B. burg-
dorferi during a heartworm screen with assays that include a
B. burgdorferi ELISA, a urinalysis could be offered in order
to assess for proteinuria.34 If proteinuria is detected, further
work-up that includes a urine protein to creatinine ratio, aero-
bic bacterial culture of the urine, serum biochemistry panel,
and imaging may be indicated. However, whether treatment
of seropositive, proteinuric dogs with doxycycline influences
the progression of Lyme nephropathy is not known, so this
is controversial. Similarly, a CBC or platelet count could be
performed, but whether treatment of thrombocytopenic dogs
is necessary is also unknown. At a minimum, tick control for
seropositive, apparently healthy dogs should be recommended.
Public Health Aspects
Lyme disease is the most common vector-borne disease of
humans in the Northern Hemisphere, and the prevalence of the
disease has increased progressively.69 In humans, the first sign
of disease in 80% to 90% of infected individuals is erythema
migrans, which is a characteristic “bull’s-eye” rash that occurs
3 to 30 days after a tick bite and moves outward from the bite
site at a rate of approximately 1 cm/day. In some people, the
rash is pruritic or painful, and it may be accompanied by head-
ache and malaise. Early disseminated disease can appear as mul-
tiple erythema migrans rashes; myocardial disease that is usually
characterized by atrioventricular block; or neuroborreliosis.
Neuroborreliosis is more common in Europe and may be char-
acterized by the development of cranial nerve palsies or signs of
meningitis and polyradiculoneuritis. Arthritis is a manifestation
of late Lyme disease. A small percentage of infected individuals,
especially those of certain human leukocyte antigen (HLA) types,
can develop chronic, antibiotic-refractory arthritis, which may
result from persistence of spirochete residues in tissues.70 Euro-
pean strains, particularly B. afzelii, can also induce a chronic skin
manifestation known as acrodermatitis chronica atrophicans.71
Dogs do not pose a direct zoonotic risk to humans in the
household. However, they may carry infected, unfed ticks
into the household that could subsequently attach to humans.
The presence of antibodies in dogs can also indicate increased
human risk as a result of common exposure to infected ticks in
the environment (sentinel exposure).
 CHAPTER 51  Lyme Borreliosis 495

CASE EXAMPLE Neutrophils 15,561 cells/µL (3000-10,500 cells/µL)

Lymphocytes 171 cells/µL (1000-4000 cells/µL)
Monocytes 1026 cells/µL (150-1200 cells/µL)
Signalment: “Dako”, a 7-year-old MC Doberman Pinscher dog Eosinophils 342 cells/µL (0-1500 cells/µL), platelets clumped.
from Amador County in northern California Serum Chemistry Profile:
History: Dako was evaluated for a 2-day history of lethargy, Sodium 148 mmol/L (143-151 mmol/L)
apparent blindness, inappetence, and inability to walk. He Potassium 3.5 mmol/L (3.6-4.8 mmol/L)
was taken to a local emergency clinic within 24 hours of Chloride 115 mmol/L (108-116 mmol/L)
illness, where a neurologic examination was considered Bicarbonate 20 mmol/L (20-29 mmol/L)
abnormal and an intracranial lesion was suspected. He was Phosphorus 3.1 mg/dL (2.6-5.2 mg/dL)
treated with intravenous fluids and given a single dose of Calcium 10.0 mg/dL (9.6-11.2 mg/dL)
ampicillin (1 g). The following day his mentation and thoracic BUN 11 mg/dL (11-33 mg/dL)
limb strength had improved, but he remained unable to Creatinine 0.8 mg/dL (0.8-1.5 mg/dL)
walk and developed urinary incontinence. He lived on a Glucose 102 mg/dL (86-118 mg/dL)
farm and had contact with sheep, horses, llamas, chickens, Total protein 6.1 g/dL (5.4-6.9 g/dL)
geese, cats, and four other dogs, all of which were currently Albumin 3.6 g/dL (3.4-4.3 g/dL)
healthy, although an additional dog had died of acute Globulin 2.5 g/dL (1.7-3.1 g/dL)
renal failure 1 month earlier. There was no travel history or ALT 17 U/L (21-72 U/L)
toxin exposure, but frequent exposure to ticks occurred. AST 36 U/L (20-49 U/L)
Dako’s diet consisted of commercial dry dog food, and ALP 60 U/L (14-91 U/L)
occasionally pieces of cooked steak and raw goose eggs. He Creatine kinase 217 U/L (55-257 U/L)
had not received a Lyme vaccine in the past but had been GGT 1 U/L (0-6 U/L)
vaccinated regularly for distemper, adenovirus, parvovirus, Cholesterol 167 mg/dL (139-353 mg/dL)
parainfluenza, and rabies. Dako had been diagnosed several Total bilirubin 0.1 mg/dL (0-0.2 mg/dL)
years previously with color dilution alopecia. Magnesium 1.8 mg/dL (1.9-2.5 mg/dL).
Current Medications: Monthly topical flea and tick Urinalysis: SGr 1.018; pH 8.0, negative protein (SSA), negative
preventative (fipronil and S-methoprene), monthly oral bilirubin, negative glucose, 0-1 WBC/HPF, 0-2 RBC/HPF.
heartworm preventative (ivermectin and pyrantel). Imaging Findings:
Physical Examination: Thoracic Radiographs: Unremarkable.
Body Weight: 43.1 kg Abdominal Ultrasound: The liver was diffusely hypoechoic but
General: Quiet, alert, and responsive. Hydrated. Ambulatory was normal in size. There was mild bilateral adrenomegaly
on all four limbs but appeared very painful. T = 101.9°F (0.9 cm). The urinary bladder was very large and was in a
(38.8°C), HR = 78 beats/min, RR = 24 breaths/min, mucous pelvic location.
membranes pink, CRT = 1 s. Microbiologic Testing: 4Dx SNAP test (IDEXX Laboratories):
Integument, Eyes, Ears, Nose, and Throat: A thin haircoat Positive for B. burgdorferi antibodies; negative for antibodies
with scaling was noted. Mild episcleral injection was present to Anaplasma spp. and E. canis; negative for Dirofilaria
bilaterally. Moderate dental calculus and gingivitis were also immitis antigen.
present.
Musculoskeletal: Body condition score was 5/9 and the dog Cytology of Synovial Fluid Obtained via Arthro­centesis
was symmetrically well muscled. Multiple distal joints were
severely swollen and painful on palpation, including both Right Left Right Left
carpi, tarsi, stifle, and elbow joints. The dog appeared painful Carpus Carpus Tarsus Stifle
when rising from recumbency and when walked, with
pronounced right pelvic limb lameness. Cell count 23,730 29,370 ND 1020
Cardiovascular, Respiratory, Gastrointestinal, and (cells/µL)
Genitourinary: No clinically significant abnormalities were Neutrophils 85 94 76 12
detected. The dog had a large bladder, and actively urinated (%)
a large amount during the examination. Rectal examination Small mono- 2 0 1 41
was unremarkable. nuclear
Lymph Nodes: Bilateral popliteal lymphadenomegaly (3 cm in cells (%)
diameter) was noted. The remaining peripheral lymph nodes
measured 2 cm in diameter. All nodes were soft on palpation. Large mono- 13 6 23 47
Neurologic Examination: No neurologic abnormalities were nuclear
detected. cells (%)
Laboratory Findings: Interpreta- Marked purulent inflam- Very mild puru-
CBC: tion mation with nondegen- lent inflam-
HCT 48.4% (40%-55%) erate neutrophils mation with
MCV 69.6 fL (65-75 fL) nondegenerate
MCHC 34.9 g/dL (33-36 g/dL) neutrophils
WBC 17,100 cells/µL (6000-13,000 cells/µL)
ND, not done.
496 SECTION 2  Bacterial Diseases
Lyme C6 Quant antibody ELISA (IDEXX Laboratories): 153 U/mL
(normal, <30 U/mL).
PCR for B. burgdorferi on synovial fluid: Negative.
Diagnosis: Neutrophilic polyarthritis; possibly secondary to
B. burgdorferi infection.
Treatment: Dako was treated with doxycycline (5 mg/kg PO
q12h) and tramadol (2 mg/kg PO q8h) and showed dramatic
clinical improvement within 24 hours of initiating treatment.
A physical examination was normal 2 weeks later.
Comments: The positive serology and response to
doxycycline suggested the possibility of Lyme arthritis,
although it is possible there was some other cause that was
not identified and the presence of C6 antibodies was not
related to this dog’s illness. It may have been too early in
SUGGESTED READINGS
Little SE, Heise SR, Blagburn BL, et  al. Lyme borreliosis in dogs and
humans in the USA. Trends Parasitol. 2010;26(4):213-218.
Littman MP, Goldstein RE, Labato MA, et  al. ACVIM small animal
consensus statement on Lyme disease in dogs: diagnosis, treatment,
and prevention. J Vet Intern Med. 2006;20(2):422-434.
Radolf JD, Caimano MJ, Stevenson B, et  al. Of ticks, mice and men:
understanding the dual-host lifestyle of Lyme disease spirochaetes.
Nat Rev Microbiol. 2012;10(2):87-99.
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CHAPTER 52
Bartonellosis
Jane E. Sykes and Bruno B. Chomel
Overview of Bartonellosis in Dogs and Cats
First Described: Peru, 1905 (Alberto Barton Thompson) (Bar-
tonella bacilliformis).1 Identification of Bartonella as the
cause of cat scratch disease in humans did not occur until
1983.2
Causes: The most common species in cats are Bartonella
henselae and Bartonella clarridgeiae; in dogs, they are Bar-
tonella vinsonii subsp. berkhoffii and B. henselae.
Geographic Distribution: Worldwide, with highest preva-
lence in subtropical and tropical regions
Major Mode of Transmission: Fleas (Ctenocephalides felis);
possibly other flea species (such as Pulex spp.) and vectors
such as ticks, lice, biting flies
Major Clinical Signs: Signs that relate to infective endocar-
ditis (lethargy, fever, cardiac murmur, cough, tachypnea,
lameness, neurologic signs) are the most frequent and
well understood manifestations of bartonellosis in dogs.
Bartonella can also cause endocarditis and myocarditis in
cats and is suspected to cause (or contribute to) caudal
stomatitis and possibly other systemic inflammatory dis-
orders such as uveitis.
Differential Diagnoses: Cats: major differential diagnoses are
other bacterial causes of endocarditis and feline infec-
tious peritonitis. Dogs: other bacterial causes of endo-
carditis, other vector-borne diseases such as ehrlichiosis,
anaplasmosis, borreliosis and babesiosis, and immune-
mediated inflammatory diseases.
Human Health Significance: Bartonella causes cat scratch
disease in immunocompetent humans as well as vasculo-
proliferative disorders in immunocompromised humans.
Etiology and Epidemiology
Bartonella spp. are fastidious, intraerythrocytic gram-negative
bacteria that infect a wide range of domestic and wild mamma-
lian host species. Different Bartonella species have adapted to
specific mammalian reservoir hosts and can infect and occasion-
ally cause disease in alternative, incidental hosts. More than 10
species of Bartonella infect cats or dogs worldwide (Tables 52-1
and 52-2). Bartonella infections are important because cats are
the principal reservoir host for Bartonella henselae, the main
cause of cat scratch disease (CSD) in humans, and subclinical
bacteremia in cats is widespread (8% to 56% of healthy cats
worldwide).3
498
Cat fleas (Ctenocephalides felis) play a major role in the
transmission of feline Bartonella infections. The presence of cat
fleas is essential for maintenance of the infection within the cat
population.4 B. henselae can multiply in the digestive system of
the flea and survive several days in flea feces.5 The main source
of infection appears to be flea feces that are inoculated by con-
taminated cat claws.6 There is some evidence that Bartonella
is present in the saliva of cats, but shedding of B. henselae in
cat saliva has not been clearly documented. Other potential
vectors, such as Pulex flea species, ticks, lice, and biting flies,
also harbor Bartonella DNA, and experimental transmission of
Bartonella birtlesii has been accomplished with Ixodes ricinus
ticks.7 There is epidemiologic evidence that ticks may transmit
­Bartonella to dogs.
Because Bartonella infections are often subclinical in dogs
and cats, the full extent to which Bartonella spp. cause disease
in naturally infected dogs and cats remains unclear. Bartonella
spp. have been widely studied for their role in a number of
idiopathic inflammatory disorders of cats, including uveitis,
lymphadenopathy, caudal stomatitis, and rhinitis, without
definitive evidence of disease causation. Without doubt, they
are important causes of endocarditis in dogs and occasionally
cause endocarditis and myocarditis in cats (Table 52-3 and
Box 52-1).
Epidemiology of Bartonella in Cats
The most common Bartonella species isolated from cats is B.
henselae. Less commonly, cats are infected with Bartonella clar-
ridgeiae or Bartonella koehlerae, which also have been linked
to human disease.5,8 Young cats (≤1 year) are more likely than
older cats to be bacteremic, and stray or feral cats are more
likely to be bacteremic than pet cats.5,9,10 Older cats are more
likely to be seropositive than young cats, which likely reflects
the increased chance of exposure over time. The prevalence of
Bartonella bacteremia in cats is also highest in warm, humid cli-
mates (e.g., 68% in the Philippines), whereas it is low in colder
climates (e.g., 0% in Norway).5 At least two genotypes of B.
henselae infect cats, type Houston-1 (type I) and type Marseille
(type II). B. henselae type Marseille predominates among cats
in the western United States, western continental Europe, the
United Kingdom, and Australia; type Houston-1 is dominant
in Asia (Japan and the Philippines).5 However, within a given
country, the prevalence of these types varies among cat popula-
tions, and type Houston-1 is more often isolated from humans
with bartonellosis, even when type Marseille is more prevalent
in the cat population. Some molecular typing methods reveal an
even broader genetic diversity among feline strains, and most
of the strains that infect humans cluster in a limited number of
groups.
 CHAPTER 52  Bartonellosis 499

TABLE 52-1 TABLE 52-3

Species of Bartonella Known to Infect Cats Examples of Clinical Manifestations That May, in Some
Circumstances, Be Associated with Bartonella Infection in
Bartonella Primary Primary Naturally Infected Cats
Species Reservoir Vector
B. henselae Cats Cat flea (Ctenocephalides Strength of Clinical Bartonella
felis) Association Manifestation Species
B. clarridgeiae Cats Cat flea Definite Endocarditis26 B. henselae
B. koehlerae Probably cats Cat flea Myocarditis and
B. quintana Human Human body louse ­diaphragmatic myositis27
­(Pediculus humanus) Probable Osteomyelitis28 B. v. berkhoffii
B. bovis Ruminants Biting flies? Systemic reactive
(cattle, deer) ­angioendotheliomatosis29
B. vinsonii Coyotes, do- Unknown (fleas, ticks?) Possible* Caudal stomatitis32-37 Unknown
subspecies mestic dogs Lower urinary tract
berkhoffii ­disease32,36
Uveitis30,31
Lymphadenopathy37
TABLE 52-2 Fever38
Species of Bartonella Known to Infect Dogs *Results of experimental studies are conflicting or there is insufficient
evidence to prove an association with disease.
Bartonella Species Primary Reservoir Primary Vector
B. henselae Domestic cats Cat flea (Ctenoce-
phalides felis) Epidemiology of Bartonella in Dogs
B. vinsonii subsp. Coyotes, domestic Unknown (fleas, Dogs are most commonly infected with Bartonella vinsonii
berkhoffii dogs, foxes ticks?) subsp. berkhoffii or B. henselae. Domestic and wild dogs are
B. rochalimae Wild carnivores, Fleas (Pulex thought to be the natural reservoir for B. v. berkhoffii, because
domestic dogs ­irritans) B. v. berkhoffii establishes prolonged bacteremia in dogs. In
California, 35% of coyotes (Canis latrans) tested were seroposi-
B. clarridgeiae Domestic cats Cat flea tive, and 28% of coyotes from a highly endemic region were
B. koehlerae Domestic cats Unknown bacteremic.5 Other Bartonella species have also been detected in
B. quintana Human Body louse (Pedic- dogs (see Table 52-2). Novel species also have been identified
ulus humanus) in dogs from Thailand, Sri Lanka, and the Mediterranean.11-13
(in humans) Because it is often difficult to culture Bartonella from domes-
tic dog blood, prevalence studies in dogs are usually based on
B. washoensis California ground Unknown
antibody detection. Unfortunately, this correlates poorly with
squirrel
bacteremia. In addition, serologic cross-reactivity occurs among
B. bovis Ruminants (cattle, Unknown Bartonella species so seroprevalence studies cannot be Barton-
deer) ella species-specific. The prevalence of antibodies to B. v. berk-
B. elizabethae Rats Oriental rat flea hoffii in dogs is highest in tropical or subtropical regions. For
(Xenopsylla example, seroprevalences as high as 47% were detected in stray
cheopis) dogs from Morocco.14 In the southeastern United States, the
B. grahamii Wild mice Rodent fleas prevalence of B. henselae antibodies in healthy dogs was 10%;
(Ctenophthal- a higher prevalence (27%) was found in sick dogs.15 Risk fac-
mus nobilis) tors identified in dogs in the southeastern United States were
tick exposure, residence in a rural environment, roaming, and
B. taylorii Wild mice Rodent fleas
outdoor exposure.16 The prevalence of B. henselae antibodies
(Ctenophthal-
in sick dogs from the western United States is less than 2%.17
mus nobilis)
B. vinsonii subsp. White-footed Unknown Clinical Features
arupensis mouse
B. volans–like Southern flying Unknown Signs and Their Pathogenesis
squirrel After infection, Bartonella replicates in erythrocytes and can also
“Candidatus Dogs, jackals Unknown infect endothelial cells and bone marrow progenitor cells.18 It
B. merieuxii” then establishes chronic, often subclinical bacteremia, which can
(strain HMD) last for weeks, months, or even more than a year and may be a
specific adaptation to a mode of transmission by blood-sucking
500 SECTION 2  Bacterial Diseases
BOX 52-1
Descriptive Case Reports or Small Case Series Where Bartonella Has Been Detected within Lesions in Dogs using Culture or PCR
Endocarditis
B. v. berkhoffii45-47
B. henselae45,48
B. clarridgeiae46
B. koehlerae48
B. quintana49
B. rochalimae50
Chronic lymphocytic hepatitis with copper
accumulation (Doberman)
B. clarridgeiae51
Systemic pyogranulomatous disease, hyperviscosity
syndrome
Bartonella spp.52
Pyogranulomatous lymphadenitis
B. henselae53
Granulomatous hepatitis
B. henselae51
Peliosis hepatis
B. henselae54
arthropods.19 Infection of endothelial cells may be more likely
to occur in incidental hosts and appears to be necessary for the
development of disorders such as vasculoproliferative disease and
endocarditis. Bartonella is thought to cause vasculoproliferative
disease via cytokine-induced stimulation of endothelial cell pro-
liferation and inhibition of endothelial cell apoptosis. Virulence
factors characterized in Bartonella species include adhesins, heme
acquisition mechanisms, type IV secretion systems, and a low-
potency lipopolysaccharide, among others.20 Host immunocom-
promise, such as due to genetic susceptibility (e.g., breed-related
immunodeficiency syndromes), poor nutrition, overcrowding,
co-infections with other pathogens, immunosuppressive drug
treatment, or defects in normal host barriers (such as congenital
valvular disease in dogs with endocarditis), may also influence
whether disease ultimately develops in infected dogs and cats.
The vast majority of infected dogs and cats show no overt
clinical signs of illness when bacteremic. In some situations, dis-
ease manifestations that occur in Bartonella spp.–infected dogs
and cats are otherwise uncommon to rare and/or known to be
associated with Bartonella infection in human patients; organ-
isms may also have been visualized with silver stains using light
microscopy in affected tissues (e.g., endocarditis or myocarditis
lesions). In other situations, nonspecific clinical signs are pres-
ent (e.g., uveitis, gingivostomatitis, chylothorax, polyarthritis,
chronic rhinitis, idiopathic lower urinary tract disease, lymph-
adenopathy, reproductive disease, or neurologic disorders), and
it has been impossible to clearly assign a role to Bartonella in
disease causation. A clear understanding of the role of Barton-
ella in disease causation has also been thwarted by limitations of
diagnostic tests available for detection of infection.
Systemic granulomatous disease and
sialometaplasia
B. henselae
B. v. berkhoffii55
Chronic erosive polyarthritis
B. henselae
B. v. berkhoffii56
Massive post-traumatic seroma
B. henselae
B. v. berkhoffii57
Hemangiopericytoma
B. v. berkhoffii or B. henselae29
Epistaxis
B. v. berkhoffii or B. henselae58
Meningoradiculoneuritis and pyogranulomatous
dermatitis or panniculitis
B. v. berkhoffii59
Bacillary angiomatosis
B. v. berkhoffii60
Clinical Manifestations in Cats
Experimental infections of specific pathogen free cats with
Bartonella have shed light on disease processes that can fol-
low infection of cats with Bartonella. Cats infected with
B. henselae develop a small papule at the site of inoculation,
and transient fever and lymphadenopathy.21-24 Transient neu-
rologic signs (such as staring and behavioral abnormalities) and
reproductive disorders can also occur. Some cats infected with
B. henselae and/or B. clarridgeiae show no clinical signs, and
gross lesions are lacking at necropsy. However, histopathology
reveals peripheral lymph node hyperplasia, splenic follicular
hyperplasia, lymphocytic cholangitis/pericholangitis, lympho-
cytic hepatitis, lymphoplasmacytic myocarditis, and/or intersti-
tial lymphocytic nephritis.21,23
In naturally infected cats, Bartonella can rarely cause endo-
carditis and myocarditis. Generally speaking, infective endocar-
ditis is rare in cats, but a few of the reported cases have been
associated with Bartonella infection. B. henselae DNA has been
amplified from aortic valvular vegetative lesions of cats, with
visualization of the organism in lesions using silver stains.25,26
Dramatic pyogranulomatous myocarditis and diaphragmatic
myositis were associated with B. henselae infection in two
cats that died in a North Carolina shelter, and organisms were
detected in lesions with silver stains and immunohistochemis-
try (IHC) (Figure 52-1).27 Carpal and metacarpal osteomyelitis
was also associated with a B. v. berkhoffii infection in a cat;
Bartonella DNA was detected in bone lesions as well as in the
blood.28 The DNA of B. v. berkhoffii and B. henselae was also
detected in cardiac tissues of several cats with systemic reactive
angioendotheliomatosis.29
 CHAPTER 52  Bartonellosis 501

B C
FIGURE 52-1  A, Granulomatous myocarditis associated with Bartonella henselae infection in an 8-week-old female domestic shorthair introduced to a shelter that was ridden
with fleas. B, Histopathology of the heart of the affected cat showing intralesional agyrophilic bacteria (arrows). Warthin-Starry stain. C, Short bacilli (arrows) in an inflammatory focus
are immunoreactive (brown) for Bartonella henselae–specific monoclonal antibody. Immunohistochemistry with diaminobenzidine chromogen, hematoxylin counterstain. (A, Courtesy
Drs. Jack Broadhurst and Edward Breitschwerdt. From Varanat M, Broadhurst J, Linder KE, et al. Identification of Bartonella henselae in 2 cats with pyogranulomatous myocarditis and
diaphragmatic myositis. Vet Pathol 2012;49:608-611.)

Bartonella has been investigated for its role in a large number
of idiopathic conditions in cats (see Table 52-3).26-38 These include
uveitis,30-32 caudal gingivostomatitis,32-37 fever of unknown
origin,38 lower urinary tract disease,32,36,39 chronic kidney dis-
ease,32,36 pancreatitis,40 lymphadenopathy (Figure 52-2),37 neu-
rologic disease,32,41,42 pododermatitis,43 and chronic idiopathic
rhinosinusitis.44 There is currently no convincing and repeatable
scientific evidence that Bartonella infection causes any of these
conditions. Studies have varied in the methodology used to detect
infection (serology, culture, and/or PCR assays), and many of the
conditions investigated likely have multifactorial etiologies. An
understanding of the significance of Bartonella in these conditions
will probably require prospective study of large numbers of cats
with clearly defined clinical illness using Bartonella culture and/
or PCR assays.
Clinical Manifestations in Dogs
Bartonella spp. are important causes of blood-culture–negative
endocarditis in dogs (see Box 52-1).25,29,45-60 Infection with
Bartonella was identified in 6 (19%) of 31 dogs with culture–
negative endocarditis in California.61 Worldwide, the DNA of
several Bartonella species has been detected in the heart valves
of dogs with endocarditis. The most common species identi-
fied has been B. v. berkhoffii. In dogs, Bartonella endocarditis
usually involves the aortic valve, although the mitral valve is
occasionally affected, so the possibility of bartonellosis should
not be ruled out in dogs with mitral valve endocarditis. Dogs
with Bartonella endocarditis are more likely to be afebrile
and more likely to have congestive heart failure than dogs
with other causes of endocarditis, and their median survival
time is shorter (Figure 52-3).61 Complications of Bartonella
endocarditis include thromboembolic disease and neutrophilic
polyarthritis.
Bartonella, or Bartonella DNA, has been detected in lesions
from dogs with a variety of chronic pyogranulomatous or gran-
ulomatous inflammatory and vasculoproliferative diseases (see
Box 52-1). Bartonella was detected in a dog with peliosis hepa-
tis and a dog with bacillary angiomatosis, rare conditions that
are strongly associated with Bartonella infection in humans.54,60
Peliosis hepatis and bacillary angiomatosis are vasculoprolifera-
tive diseases characterized by the presence of blood-filled pro-
liferations of vascular tissue in the liver and skin, respectively.
Based on case reports and seroprevalence studies, Bartonella is
suspected to cause polyarthritis, epistaxis, thrombocytopenia,
502 SECTION 2  Bacterial Diseases

and/or splenomegaly in dogs.17 However, these associations

could also reflect the presence of other detected or undetected,
co-transmitted vector-borne pathogens that cause these dis-
orders. Bartonella has been investigated for its role in canine
lymphoma62; neurologic disease63; splenic disorders including
lymphoid nodular hyperplasia, hemangiosarcoma, and fibrohis-
tiocytic nodules64; idiopathic cavitary effusion65; and idiopathic
rhinitis.66 As for cats, the role that Bartonella plays in these
conditions, if any, is unclear and requires further study.

Physical Examination Findings

FIGURE 52-2  Lymph node of a 1-year-old, retrovirus-negative, flea-ridden cat with a
3-month history of fever, mild lethargy, and generalized peripheral lymphadenopathy but
a normal appetite. The right axillary and both mandibular lymph nodes measured 3 cm in
diameter; the remaining peripheral lymph nodes were 2 cm in diameter. A serum chemis-
try profile revealed hyperglobulinemia (6.5 g/dL). Serum protein electrophoresis revealed
a polyclonal gammopathy. Histopathology of the right axillary, right mandibular and right
popliteal lymph node showed lymphofollicular hyperplasia and marked neutrophilic, lym-
phocytic, and histiocytic capsulitis and perilymphadenitis. Note the florid capsulitis to the
right of the arrows, which indicate the outer extent of the lymph node parenchyma. Silver
stains (Warthin-Starry and Steiner stains) showed a small amount of granular staining
within cells, which possibly represented intracellular agyrophilic bacteria. The DNA of Bar-
tonella henselae and Bartonella clarridgeiae was detected in the lymph node using PCR,
and blood cultures were positive for B. henselae. Interestingly, the cat was seronegative to
B. henselae and B. clarridgeiae.
In dogs with Bartonella endocarditis, physical examination
findings include lethargy, inappetence and thin body condition;
lameness or recumbency due to thromboembolic complica-
tions, weakness, or polyarthritis; joint effusion; and, uncom-
monly, fever (as high as 104.9°F [41°C]). Evidence of cardiac
disease and congestive heart failure is often present, including
respiratory distress, cough, increased lung sounds, and sys-
tolic and/or diastolic heart murmurs, cardiac arrhythmias, and
pulse deficits. Neurologic signs such as anisocoria and obtun-
dation can occur in dogs with thromboembolism that involves
the central nervous system. There may also be evidence of flea
or tick infestations in some dogs. Other physical examination
findings that might be present in dogs with other manifesta-
tions of bartonellosis are mass lesions that involve the nasal
cavity, lymph nodes, or salivary glands; splenomegaly or hepa-
tomegaly; cavitary effusions; peripheral edema; epistaxis; or
nodular skin lesions.
Physical examination findings in cats with Bartonella endo-
carditis or myocarditis have included fever, lethargy, respiratory
distress, cardiac murmurs, and arrhythmias.26,27
Diagnosis
Diagnosis of bartonellosis is based on the presence of consistent
clinical abnormalities (especially endocarditis or myocarditis,
but also other systemic inflammatory or vasculoproliferative
diseases), histopathologic findings, and positive culture or PCR

100

90

80

70 Dogs with IE caused by Bartonella spp. infection

Dogs with IE caused by other organisms
Percent survival

60

50

40

30

20

10

0
0 250 500 750 1000 1250 1500 1750 2000
Time (days)
FIGURE 52-3  Kaplan-Meier survival curve comparing survival of dogs with Bartonella infectious endocarditis (IE) with that of dogs with IE caused by other pathogens. (From Sykes
JE, Kittleson MD, Pesavento PA, et al. Evaluation of the relationship between causative organisms and clinical characteristics of infective endocarditis in dogs: 71 cases (1992-2005). J Am
Vet Med Assoc 2006;228:1723-1734.)
 CHAPTER 52  Bartonellosis 503

TABLE 52-4
Diagnostic Assays Available for Bartonella Infection in Dogs and Cats
Assay Specimen Type Target Performance
Culture Whole blood, tissue speci- Bartonella spp. Confirms infection and allows species identification. Special-
mens obtained at nec- ized culture conditions required; best performed in labo-
ropsy (e.g., heart valve, ratories with special expertise. Requires several weeks’
myocardium) or by incubation. Sensitivity is especially low in dogs because
biopsy (e.g., liver biopsy, of low-level or intermittent bacteremia. Use of BAPGM
skin biopsy) enrichment media followed by PCR may increase sensitiv-
ity but may increase the chance of false-positive results
due to PCR-related contamination. Positive culture results
alone do not apply disease causation, because healthy
dogs and cats can have Bartonella in their blood.
Histopathology with Tissue specimens obtained Bartonella spp. Low sensitivity, but supports disease causation when
organism ­detecting by biopsy or necropsy organisms positive and properly performed and interpreted.
using silver stains
or IHC
Serology Serum Antibodies to Variable cross-reactivity between Bartonella species;
Bartonella spp. species-specific assays are required. Immunofluorescent
antibody, ELISA, and Western immunoblot assays are
available. Results correlate poorly with bacteremia.
Positive results do not imply disease causation, because
a significant proportion of healthy dogs and cats are
seropositive. Positive titers are usually present in dogs
and cats with endocarditis.
PCR Whole blood, splenic or Bartonella spp. Confirms active infection more rapidly than culture.
lymph node aspirates, DNA Sensitivity and specificity may vary depending on assay
tissue specimens obtained design and specimen type; may not necessarily be more
at necropsy or biopsy sensitive than culture. Because healthy animals may be
PCR positive, positive PCR results must be interpreted
in light of the clinical signs. False positive results may
occur as a result of PCR-related contamination.

assay results on blood and affected tissues (Table 52-4). It may
be impossible to identify Bartonella as the cause of disease if
the organism is detected only in the blood and if affected tis-
sues (e.g., valvular vegetations) cannot be obtained for analy-
sis, because healthy dogs and especially cats can be bacteremic.
Other possible causes of disease must also be investigated, and
a response to antibiotic treatment could be evaluated (see Case
Example).67 It is even more difficult to establish a definitive
diagnosis based on serology, but when culture and PCR assay
results are negative, positive serologic test results in dogs may
be of some value in the presence of consistent clinical abnor-
malities. More specifically (and as occurs in humans), dogs with
Bartonella endocarditis usually have high antibody titers to
Bartonella spp. Organism detection tests are insensitive in dogs
because dogs tend to have very low levels of bacteremia when
compared with cats.
Laboratory Abnormalities
Hematologic and biochemistry abnormalities in dogs and cats
with Bartonella endocarditis are nonspecific and often mild.
The CBC may show a mild nonregenerative anemia (31% to
39%), variable leukocytosis primarily due to neutrophilia (up
to 43,000 neutrophils/µL), and mild thrombocytopenia (usually
above 100,000 platelets/µL). In a few dogs, low to moderate
numbers of circulating band neutrophils, lymphocytosis, and/
or monocytosis are present.49,68,69 The biochemistry panel
may reveal mild azotemia (creatinine <3 mg/dL), mild hypoal-
buminemia, and, less often, hyperglobulinemia. Isosthenuria,
proteinuria, cylindruria, and slightly increased urine protein-to-
creatinine ratios have been present in the urine of some affected
dogs. Dogs with hepatic disorders associated with Bartonella
infection have had moderately increased activities of serum ALT
and/or ALP.51,54
Diagnostic Imaging
Plain Radiography
Thoracic radiographs in dogs with Bartonella endocarditis
often show evidence of congestive heart failure, with venous
engorgement and pulmonary edema (Figure 52-4). Lung lobe
consolidation and hilar lymphadenopathy were described in a
dog with systemic granulomatous disease and sialometaplasia.55
Abdominal Ultrasound
Ultrasound of dogs with systemic granulomatous or pyogranu-
lomatous disease may reveal lymphadenomegaly and organo-
megaly,52 and mixed echogenic patterns within the liver.51 The
dog with peliosis hepatis had ascites and multiple, small nodular
masses and hypoechoic cystic lesions within the liver.54
504 SECTION 2  Bacterial Diseases
A
B
FIGURE 52-4  Lateral thoracic radiograph from a 3-year-old female spayed Dober-
man pinscher with early congestive heart failure due to endocarditis, before (A) and after
(B) treatment with furosemide. The dog was seroreactive to Bartonella vinsonii subsp.
berkhoffii with a titer of 1:256 and Bartonella henselae with a titer of 1:64. A, There is mild
cardiomegaly with left atrial enlargement. There is increased pulmonary interstitial opac-
ity that is most pronounced in the accessory lung lobe, as well as cranial to the heart.  “Mercedes Benz” sign has been destroyed by the aortic valvular thickenings.
B, Pulmonary infiltrates have significantly resolved.
Echocardiography
Echocardiography in dogs and cats with Bartonella endocar-
ditis reveals oscillating valvular vegetations or thickened and
hyperechoic valve leaflets, which usually involve the aortic valve
(Figure 52-5). There may be evidence of aortic regurgitation or
insufficiency and eccentric hypertrophy of the left ventricle in
some dogs.
Microbiologic Tests
Culture
Culture of blood or other tissues for Bartonella allows defini-
tive diagnosis of infection. However, blood culture requires spe-
cial techniques and can be insensitive because of the relapsing
nature of bacteremia. In contrast to the situation in cats, culture
has especially low sensitivity in dogs because of the low level
of bacteremia in dogs (especially when they act as incidental
hosts and develop disease). Blood for culture should be obtained
using sterile technique and the blood placed in EDTA-containing
A
B
FIGURE 52-5  Echocardiogram images from a 13-year old German shepherd dog
with blood culture–negative endocarditis. The dog also had biopsy-confirmed glomerulo-
nephritis. IFA serology for Bartonella vinsonii subsp. berkhoffii, Bartonella clarridgeiae, and
Bartonella henselae were positive at titers of 1:512, 1:1024, and 1:128, respectively. On the
long-axis view (A) there is an irregular hyperechoic lesion attached to the aortic valve
cusps (arrow), and the cusps appear thickened. On the short-axis view (B), the normal
tubes, and then chilled or frozen during shipment. Collection
of blood into plastic EDTA tubes allows the tubes to be fro-
zen directly, which releases organisms from erythrocytes after
which they are plated onto special media. Blood should be
sent to laboratories familiar with the culture of these fastidious
organisms, and the laboratories should be contacted for specific
instructions regarding specimen collection and submission. Spe-
cial media (such as fresh chocolate agar, or brain-heart infusion
agar enriched with 5% to 10% blood) and culture conditions
(5% CO2, temperatures of 35°C to 37°C) are used to enhance
the likelihood of isolation of Bartonella.62 Extended incubation
times of up to 6 to 8 weeks are often required. Serial Bartonella
blood cultures may enhance the chance of a positive result. The
use of an insect growth medium (Bartonella Alpha Proteobacte-
ria Growth Medium, or BAPGM) that is enriched with multiple
micronutrients and defibrinated sheep blood for pre-enrichment
culture of blood and potentially other tissues, followed by PCR
assay of the liquid culture and subinoculation of the liquid cul-
ture onto agar plates, may enhance the sensitivity of culture for
 CHAPTER 52  Bartonellosis 505

diagnosis of Bartonella infections of dogs. It also has the poten-

tial to yield other fastidious organisms not cultured under rou-
tine conditions that may play a role in endocarditis.70

Molecular Diagnosis Using the Polymerase Chain Reaction

A variety of PCR-based assays have been developed for rapid
detection of Bartonella DNA in blood or other tissues. Some
veterinary diagnostic laboratories offer real-time PCR assays
for detection of Bartonella species as a component of vector-
borne PCR panels. Some assays detect all species of Bartonella,
whereas others are species-specific. Genetic analysis of the resul-
tant PCR products can also be performed to identify the infect-
ing species. The clinical performance of many of these assays
when compared with culture has not been well studied. When
used directly on blood specimens, PCR assays may be no more
sensitive than culture for detection of Bartonella, and detection
of DNA does not always equate to detection of living organ-
isms. False-positive PCR assay results due to laboratory con- A
tamination have the potential to occur.

Serologic Diagnosis
Immunofluorescent antibody (IFA), ELISA, and Western immu-
noblot assays are available for detection of antibodies to Bartonella
species. Some degree of cross-reactivity occurs among Bartonella
species, but multiple species-specific assays still should be per-
formed to increase the chance of antibody detection. Paired titers
are not valuable because disease typically results from chronic and
persistent infection. If serologic testing is performed in dogs, at a
minimum it should include testing for B. henselae, B. v. berkhoffii,
B. rochalimae, and B. clarridgeiae. Measurement of serum anti-
bodies to Bartonella has a limited ability to help determine whether
a sick animal has an active Bartonella infection. In both cats and
dogs, bacteremia can occur in the absence of detectable antibodies
(see Figure 52-2), and seropositive cats and dogs are not always
bacteremic. Fewer than half of antibody-positive cats are bactere-
mic, and 3% to 15% of antibody-negative cats are bacteremic.67
Because of this, serology is not recommended for routine diagnosis
of Bartonella infection in cats. Because of the low sensitivity of
PCR and culture in dogs, serology could be considered when these B
assays and routine blood cultures are negative (or in conjunction FIGURE 52-6  Gross pathologic findings (A) and histopathology (B) of the aortic
with PCR and culture) to support a diagnosis of Bartonella endo- valve from a dog with Bartonella endocarditis. A, The periphery of the valve is eroded. Vari-
carditis (which is usually associated with high antibody titers). ably sized, mineralized nodules expand the valve leaflet. There are multifocal, slightly raised,
dull white plaques on the surface of the adjacent aorta and ventricular endocardium. B, Aor-
Pathologic Findings tic valve, aorta, and ventricular septum. The distal leaflet is fragmented and contains mineral 
Gross necropsy of dogs and cats with Bartonella endocarditis deposits (arrow) and hemorrhage. At the base of the valve, the endothelial surface is lifted
and there are broad bands of loose connective tissue, small vessels, and endothelial-lined
may reveal evidence of congestive heart failure, with lung con-
clefts (arrowheads). H&E stain. Bar = 180 µm. (Reprinted from Pesavento PA, Chomel BB, Kas-
gestion and edema. Single or multiple, variably sized nodules are ten RW, et al. Pathology of Bartonella endocarditis in six dogs. Vet Pathol 2005;3:370-373.)
typically present on the periphery of valvular leaflets, which may
be small and firm or large, granular, and friable (Figure 52-6).
White plaques may be found adherent to the adjacent endo- scattered through other parts of the liver. Nodular granuloma-
cardial surfaces. Histopathology reveals chronic inflammation, tous or pyogranulomatous lesions in lymphoid or other tissues
with fibrous tissue and extensive mineral deposits. The presence (such as the liver, lymph nodes, and nasal cavity) have also been
of valvular mineralization should raise suspicion for Bartonella described in dogs with bartonellosis,47,51,71 and nodular pyo-
endocarditis, because mineralization is uncommonly associated granulomatous inflammatory lesions with intralesional bacteria
with endocarditis caused by other bacteria.61 Small bacilli may were described in the myocardium and diaphragm of cats with
be seen within lesions with silver stains or immunohistochemical Bartonella myocarditis and diaphragmatic myositis.27
stains for B. henselae.26,27,60 Silver stains must be interpreted with
caution, because necrotic debris and nuclear material can mimic Treatment and Prognosis
positive staining. Gross lesions in the dog with peliosis hepatis
consisted of multiple, 0.1- to 0.3-cm cysts in the liver that were Efficacy has not been established for any antimicrobial that could
filled with cloudy, serosanguineous fluid.54 The cysts were lined be used to eliminate Bartonella bacteremia in cats or dogs. In
with endothelial cells, and there was evidence of telangiectasia humans, doxycycline, erythromycin, and rifampin are the most
506 SECTION 2  Bacterial Diseases

TABLE 52-5
Medications That Could Be Used to treat Bartonella spp.
Infections in Sick Dogs and Cats Suspected to have Bartonellosis
Dose Interval
Drug (mg/kg) Route (hours)
Ampicillin 10-20 IV 6-8
sodium
Gentamicin 9-14 (dogs), IV, IM, 24
sulfate* 5-8 (cats) SC
Amoxicillin- 12.5-25 PO 8
clavulanate
Doxycycline 10 PO 12
Rifampin† 5 PO 12
Azithromycin 5-10 PO 24
Marbofloxacin 2.75-5.5 PO 24
Enrofloxacin‡ 5 (cats), PO 24
5-20 (dogs)

*Use in combination with ampicillin for initial treatment of endocardi-

tis; other aminoglycosides could be substituted for gentamicin (such as
amikacin). Monitor renal values during treatment.
†Use in combination with doxycycline; avoid monotherapy with ri-

fampin because of the potential for rapid selection for resistant bacteria.
May cause gastrointestinal adverse effects.
‡Other fluoroquinolones are preferred in cats due to the higher risk of
retinal toxicity with enrofloxacin. Doses of 5 mg/kg q24h should never
be exceeded in cats.
frequently recommended antimicrobials for treatment of Barton-
ella infection, but clinical improvement has been reported after
the use of penicillin, gentamicin, ceftriaxone, ciprofloxacin, or
azithromycin. Treatment for 2 weeks is recommended for immu-
nocompetent patients (e.g., for CSD), and for a minimum of 6
weeks in immunocompromised humans. Relapses in bacteremia,
fever, or other disease manifestations have been reported in immu-
nocompromised humans, despite a 6-week treatment regimen.
The prognosis for Bartonella endocarditis in dogs and cats is
generally poor. Supportive care and medications such as furo-
semide to manage congestive heart failure are often required. A
combination of a β-lactam and an aminoglycoside or doxycy-
cline is recommended for treatment of Bartonella endocarditis
in human patients, and valve replacement is usually indicated
(see Chapter 86).72 Oral antimicrobial alternatives for dogs that
are stable include high-dose doxycycline, alone or in combina-
tion with rifampin; azithromycin; or fluoroquinolones, alone or
in combination with amoxicillin (Table 52-5). Resistance of B.
henselae to azithromycin and fluoroquinolones has been docu-
mented.73 Data from experimentally or naturally infected cats
indicate that a high dose of doxycycline may be necessary to elim-
inate Bartonella infection in cats. Amoxicillin–clavulanic acid
may also be an appropriate choice for treatment of infected cats
when a definitive diagnosis is not known. If there is no response
after 7 days, a change to a fluoroquinolone or azithromycin could
be considered.67 Extreme care should be taken to avoid scratches
or bites when administering drugs to bacteremic cats.
Immunity and Vaccination
There are no vaccines for prevention of bartonellosis in cats or
dogs. Bartonella evades the host immune system and establishes
FIGURE 52-7  A crop of cutaneous bacillary angiomatosis lesions on the elbow of an
AIDS patient. All lesions involuted with doxycycline treatment. (From Slater LN, Welch DF.
Bartonella, including cat-scratch disease. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell,
Douglas and Bennett’s Principles and Practice of Infectious Diseases, 7 ed. Philadelphia, PA:
Churchill Livingstone Elsevier; 2010:2995-3009.)
a persistent intracellular infection. Dogs and cats can be co-
infected with multiple different Bartonella species and geno-
types. Vaccination and challenge with the same or different
Bartonella species results in variable protection, and protection
is generally species specific.74-76
Prevention
Prevention of Bartonella infections in cats and dogs is best
accomplished by avoiding exposure to arthropod vectors. Flea
control measures, such as use of ectoparasiticides, can interrupt
transmission of B. henselae among cats and should be main-
tained year-round.67,77 Housing cats indoors may also reduce
exposure to fleas. B. henselae and B. clarridgeiae have been
transmitted through inoculation of infected cat blood; therefore,
cats of unknown Bartonella status or cats that are positive by
culture, serology, and/or PCR for Bartonella should be excluded
as blood donor cats.
Public Health Aspects
Cats are the major reservoirs for B. henselae, the principal
cause of CSD in immunocompetent humans. CSD is char-
acterized by formation of a pustule at the site of inoculation
(a cat scratch) within 14 days, followed by development of local
lymphadenopathy and malaise 1 to 3 weeks after the scratch (or
bite), which can persist for weeks to months. Most cases of CSD
are self-limiting after several months and respond minimally to
antimicrobial drug treatment. Occasionally CSD is complicated
by other disorders such as encephalopathy, relapsing bactere-
mia, osteomyelitis, neuroretinitis, or endocarditis, which require

antimicrobial drug treatment. Vasculoproliferative diseases such
as bacillary peliosis (e.g., peliosis hepatis) and bacillary angioma-
tosis (Figure 52-7) are among the most common complications
in immunocompromised humans.20 Young cats are most often
implicated in transmission to humans, because young cats are
more likely to be bacteremic than older cats. Dogs are less likely
to act as reservoirs for human disease when compared with cats,
because the prevalence of Bartonella infection and the magni-
tude of bacteremia in dogs are lower than that in cats. Neverthe-
less, dogs have occasionally been implicated in transmission of
Bartonella to humans through bite wounds or scratches, and
to date, all Bartonella species identified in sick dogs are also
pathogenic or potentially pathogenic in humans. Immunocom-
promised humans should preferably choose adult, well-social-
ized cats over kittens as pets and should be properly educated
in regard to the need for flea prevention. Cats’ claws should be
trimmed regularly, but there is no evidence that declawing pre-
vents transmission. Animal scratches or bites should be avoided,
and if they occur, they should be washed thoroughly with soap
and water, after which prompt medical attention should be
sought. The Guidelines for Preventing Opportunistic Infections
among HIV-Infected Persons (United States Public Health Ser-
vice and Infectious Diseases Society of America) state “No evi-
dence indicates any benefits to cats or their owners from routine
culture or serologic testing of the pet for Bartonella infection.”78
There is also no antimicrobial drug treatment that reliably elimi-
nates bacteremia from cats, and administration of antimicrobial
drugs to cats may result in unnecessary bites, scratches, and con-
tact with cat saliva. Pros and cons for the testing of healthy cats
for Bartonella infection are shown in Box 52-2.67
Bartonella has the potential to be transmitted to veterinar-
ians as a result of scratches or bite wounds as well as needle-
stick injuries.79,80 Because of the high prevalence of Bartonella
bacteremia in stray and young cats, veterinarians should take
care to avoid these injuries (consider use of sedation whenever
possible if necessary), thoroughly wash wounds that occur,
and seek medical attention early if necessary. Gloves should be
worn if skin contact with animal blood or saliva is anticipated.
Although cats and dogs are important reservoirs of Bar-
tonella, humans can also develop bartonellosis through other
routes of transmission, such as via other arthropods and
CASE EXAMPLE
Signalment: “Pesto,” a 16-month-old female spayed domestic
medium hair cat from Rio Vista in northern California
History: Pesto was evaluated at the University of California,
Davis, Veterinary Medical Teaching Hospital for progressive
lethargy over the previous month. Since obtained as a kitten,
she had shown intermittent signs of upper respiratory tract
disease, characterized by sneezing, serous to mucopurulent
nasal and ocular discharges, inappetence, blepharospasm,
tachypnea, and a cough, for which she had been treated
with amoxicillin or doxycycline on at least three separate
occasions. Between episodes she appeared healthy, but
she had always been thin. There had been no vomiting,
diarrhea, or changes in thirst or urination. Pesto was obtained
as a stray kitten, tested negative for FeLV and FIV, and had
CHAPTER 52  Bartonellosis 507
BOX 52-2
Advantages and Disadvantages of Routine Testing
of Healthy Cats for Bartonella spp. Infection
Advantages
Cats with positive Bartonella test results can be eliminated
as blood donors or breeding animals
Cats with negative Bartonella test results (serology and
culture or PCR) may be safer pets than cats that test
positive
Testing cats for Bartonella spp. may allow veterinarians to
avoid claims or litigation
Disadvantages
Bartonella serology and PCR can be falsely positive
Some cats with positive serologic test results may have
eliminated infection and be immune to reinfection
Cats with negative test results may still be bacteremic, or
become bacteremic within the near future
Detection of positive Bartonella spp. test results in some
situations may lead to needless euthanasia
A focus on testing may lead to underappreciation of the
need for other relevant health care such as flea control
There is no treatment that reliably eliminates bacteremia
in healthy cats, and antimicrobial drug treatment of cats
has not been shown to prevent human infection
Treatment of positive cats may increase the risk of human
infection due to an increased risk of bites and scratches
Treatment of positive cats may lead to unnecessary life-
threatening adverse drug reactions such as esophageal
strictures (doxycycline) or blindness (fluoroquinolones);
it also selects for antimicrobial-resistant bacteria
possibly through blood transfusions.81 Human body lice pri-
marily transmit B. quintana among humans (especially the
homeless), which causes relapsing fever (trench fever) and
endocarditis.
been housed indoors ever since. She was neutered at 8
months of age without complication. Five months earlier,
she traveled briefly to Oregon with the owner. She lived with
a dog and a retrovirus-negative kitten that was introduced
5 months ago. One other sibling had died of FIP, which was
confirmed on necropsy. Pesto was up to date on vaccinations
for respiratory viruses, panleukopenia virus, and rabies. She
was not receiving any medications.
Physical Examination:
Body Weight: 2.9 kg.
General: Quiet, alert, responsive, and hydrated. T = 103°F
(39.5°C), HR = 240 beats/min, RR = 40 breaths/minute, moist
and pink mucous membranes, CRT <2 s.
Integument: No ectoparasites were noted. The haircoat was
full and lustrous.
Eyes, Ears, Nose, and Throat: In the left eye, mild
conjunctivitis with moderate serous ocular discharge was
Continued
508 SECTION 2  Bacterial Diseases
present. Mild corneal opacity was present in the right eye.
No abnormalities detected on examination of the oral cavity.
There was no evidence of nasal discharge or abnormal nasal
airflow.
Musculoskeletal: Body condition score 4.5/9, ambulatory,
adequate muscle mass.
All Other Systems: No clinically significant abnormalities were
detected. A full neurologic examination was not performed.
Ophthalmologic Examination: Pesto behaved as if visual
and was relatively comfortable with mild blepharospasm.
The menace response, palpebral reflexes, and pupillary
light reflexes were normal. There was no evidence of eyelid
abnormalities or episcleral or conjunctival injection. Mild
chemosis was present bilaterally. There was no aqueous
flare, the lenses were normal, and a fundic examination was
normal. The dorsal third of the right cornea had superficial
vessels that extended from the limbus and a 2- to 3-mm
central area where ghost vessels were present, which may
have represented a previous dendritic ulcer. A rose Bengal
stain was negative for ulceration.
Laboratory Findings:
CBC:
HCT 31% (30%-50%)
MCV 37.8 fL (42-53 fL)
MCHC 34.2 g/dL (30-33.5 g/dL)
Reticulocytes 29,000 cells/µL (7000-60,000 cells/µL)
WBC 7030 cells/µL (4500-14,000 cells/µL)
Neutrophils 4570 cells/µL (2000-9000 cells/µL)
Lymphocytes 2179 cells/µL (1000-7000 cells/µL)
Monocytes 141 cells/µL (50-600 cells/µL)
Eosinophils 70 cells/µL (150-1100 cells/µL)
Basophils 70 cells/µL (0-50 cells/µL)
Platelets clumped but adequate. A few Dohle bodies were
seen within neutrophils.
Serum Chemistry Profile:
Sodium 146 mmol/L (151-158 mmol/L)
Potassium 4.5 mmol/L (3.6-4.9 mmol/L)
Chloride 110 mmol/L (117-126 mmol/L)
Bicarbonate 18 mmol/L (15-21 mmol/L)
Phosphorus 5.1 mg/dL (3.2-6.3 mg/dL)
Calcium 9.8 mg/dL (9.0-10.9 mg/dl)
BUN 20 mg/dL (18-33 mg/dL)
Creatinine 1.0 mg/dL (1.1-2.2 mg/dL)
Glucose 119 mg/dL (63-118 mg/dL)
Total protein 12.0 g/dL (6.6-8.4 g/dL)
Albumin 2.4 g/dL (2.2-4.6 g/dL)
Globulin 9.6 g/dL (2.8-5.4 g/dL)
ALT 42 U/L (27-101 U/L)
AST 25 U/L (17-58 U/L)
ALP 8 U/L (14-71 U/L)
GGT 9 U/L (0-4 U/L)
Cholesterol 134 mg/dL (89-258 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL).
Serum Protein Electrophoresis: Results were consistent with
a polyclonal gammopathy.
Imaging Findings:
Thoracic Radiographs: No clinically significant abnormalities
were noted.
Abdominal Ultrasound: Lymph nodes were moderately
enlarged throughout the abdomen, including the gastric,
splenic, and jejunal nodes. Hepatic and sublumbar nodes
were enlarged to a lesser degree. The echotexture of the
enlarged nodes was homogeneous. The left adrenal gland
was moderately enlarged, measuring 0.66 cm in thickness.
The right adrenal gland could not be differentiated from
multiple enlarged nodes. No free fluid was identified.
Microbiologic Testing: In-clinic FeLV antigen and FIV
antibody ELISA assay: Negative.
Feline coronavirus serum antibody IFA assay: Positive at
1:25,600.
Treatment: Pesto was treated with l-lysine (500 mg PO q12h),
for possible FHV-1 infection, and doxycycline (10 mg/kg
PO q24h, immediately followed by a bolus of water), for
possible chlamydiosis or mycoplasmosis. The owner could
not administer the l-lysine, only the doxycycline, and
reported improvement in Pesto’s activity level and appetite
several days after starting the doxycycline.
Additional Diagnostics and Outcome: Pesto was
evaluated again 2 weeks later for possible lymph node
aspiration or biopsy under sedation. Abdo­minal ultrasound
showed persistent abdominal lymphadenomegaly.
Cytologic examination of an ultrasound-guided aspirate
of a mesenteric lymph node revealed mild to moderate
lymphoid reactivity. Plasma cells were increased in number
and were admixed throughout the specimen with rare
mast cells and eosinophils. Low numbers of vacuolated
macrophages and nondegenerate neutrophils were also
observed. RT-PCR for feline coronavirus RNA on the lymph
node aspirate was negative. IFA serology for antibodies to B.
clarridgeiae was negative, whereas serology for antibodies
to B. henselae was positive at 1:64. B. henselae was cultured
from the blood. Treatment was changed from doxycycline
to azithromycin, and again there was mild improvement in
Pesto’s attitude. However, several days later the cat became
lethargic again, and her appetite decreased further.
After 3 weeks of treatment with azithromycin, Pesto was
reexamined. Because of the cat’s fractious temperament,
sedation was required. No abnormalities were detected
on physical examination. A CBC showed a hematocrit
of 23%, MCV of 39.2 fL, MCHC of 32.6 g/dL, reticulocyte
count of 23,200 cells/µL, 8220 slightly toxic neutrophils/
µL, 788 band neutrophils/µL, 1239 lymphocytes/
µL, 788 eosinophils/µL, and 298,000 platelets/µL.
A biochemistry panel showed similar abnormalities to those
previously present (mild hyponatremia, hypochloremia,
and hyperglycemia and low creatinine), and the globulin
had increased to 12.4 mg/dL, with a total protein of
14.4 mg/dL. IFA serology and culture for Bartonella were
repeated and showed a titer of 1:1024 to B. clarridgeiae and
1:4096 to B. henselae; Bartonella spp. culture was negative.
An ultrasound-guided biopsy of an enlarged mesenteric
lymph node was obtained. Histopathology showed a mixed,
dense inflammatory infiltrate composed of neutrophils,
lymphocytes, and plasma cells. Some of the macrophages
present stained positive using immunohistochemistry for
feline coronavirus antigen. Treatment with prednisone
(1 mg/kg PO q12h for 5 days, then 1 mg/kg PO q24h
thereafter) was initiated. Four days later, the cat became
anorectic and tachypneic and was euthanized. A necropsy
was not performed.

Diagnosis: Feline infectious peritonitis and concurrent B.
henselae bacteremia
Comments: Bartonella infection in this cat was initially
suspected because of fever and lymphadenopathy, and
infection with B. henselae was confirmed with Bartonella
culture. Ultimately, an alternate diagnosis (FIP) was made
based on the presence of the combination of marked
hyperglobulinemia, a very high feline coronavirus titer, and
histopathology of a mesenteric lymph node and IHC for
coronavirus antigen. There was also minimal response to
antimicrobial drugs with activity against Bartonella. Thus, the
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detection of Bartonella henselae and Bartonella vinsonii subsp.
berkhoffii from dogs with idiopathic cavitary effusions. J Vet Intern
Med. 2009;23:186-189.
66. Hawkins EC, Johnson LR, Guptill L, et al. Failure to identify an
association between serologic or molecular evidence of Bartonella
infection and idiopathic rhinitis in dogs. J Am Vet Med Assoc.
2008;233:597-599.
67. Brunt J, Guptill L, Kordick DL, et  al. American Association of
Feline Practitioners 2006 Panel report on diagnosis, treatment,
and prevention of Bartonella spp. infections. J Feline Med Surg.
2006;8:213-226.
68. Cockwill KR, Taylor SM, Philibert HM, et al. Bartonella vinsonii
subsp. berkhoffii endocarditis in a dog from Saskatchewan. Can
Vet J. 2007;48:839-844.
69. Chomel BB, Mac Donald KA, Kasten RW, et al. Aortic valve endo-
carditis in a dog due to Bartonella clarridgeiae. J Clin Microbiol.
2001;39:3548-3554.
70. Davenport AC, Mascarelli PE, Maggi RG, et al. Phylogenetic diver-
sity of bacteria isolated from sick dogs using the BAPGM enrichment
culture platform [Abstract]. J Vet Intern Med. 2012;26:786-787.
71. Pappalardo BL, Brown T, Gookin JL, et al. Granulomatous disease
associated with Bartonella infection in 2 dogs. J Vet Intern Med.
2000;14:37-42.
72. Gould FK, Denning DW, Elliott TS, et al. Guidelines for the diag-
nosis and antibiotic treatment of endocarditis in adults: a report of
the Working Party of the British Society for Antimicrobial Chemo-
therapy. J Antimicrob Chemother. 2012;67:269-289.

73. Biswas S, Maggi RG, Papich MG, et al. Molecular mechanisms of
Bartonella henselae resistance to azithromycin, pradofloxacin and
enrofloxacin. J Antimicrob Chemother. 2010;65:581-582.
74. Yamamoto K, Chomel BB, Kasten RW, et  al. Homologous pro-
tection but lack of heterologous-protection by various species and
types of Bartonella in specific pathogen-free cats. Vet Immunol
Immunopathol. 1998;65:191-204.
75. Greene CE, McDermott M, Jameson PH, et al. Bartonella henselae
infection in cats: evaluation during primary infection, treatment,
and rechallenge infection. J Clin Microbiol. 1996;34:1682-1685.
76. Regnery RL, Rooney JA, Johnson AM, et  al. Experimentally
induced Bartonella henselae infections followed by challenge exposure
and antimicrobial therapy in cats. Am J Vet Res. 1996;57:1714-1719.
77. Bradbury CA, Lappin MR. Evaluation of topical application of 10%
imidacloprid–1% moxidectin to prevent Bartonella henselae trans-
mission from cat fleas. J Am Vet Med Assoc. 2010;236:869-873.
CHAPTER 52  Bartonellosis 511
78. Kaplan JE, Masur H, Holmes KK. Guidelines for preventing oppor-
tunistic infections among HIV-infected persons—2002. Recom-
mendations of the U.S. Public Health Service and the Infectious
Diseases Society of America. MMWR Recomm Rep. 2002;51:1-52.
79. Oliveira AM, Maggi RG, Woods CW, et al. Suspected needle stick
transmission of Bartonella vinsonii subspecies berkhoffii to a vet-
erinarian. J Vet Intern Med. 2010;24:1229-1232.
80. Lin JW, Chen CM, Chang CC. Unknown fever and back pain
caused by Bartonella henselae in a veterinarian after a needle punc-
ture: a case report and literature review. Vector Borne Zoonotic
Dis. 2011;11:589-591.
81. Magalhaes RF, Urso Pitassi LH, Lania BG, et al. Bartonellosis as
cause of death after red blood cell unit transfusion. Ultrastruct
Pathol. 2009;33:151-154.
CHAPTER 53
Canine Brucellosis
Autumn P. Davidson and Jane E. Sykes
Overview of Brucellosis in Dogs
First Described: B. canis was first isolated from dogs by Leland
Carmichael in 1966 (USA)1
Causes: Brucella canis; rarely Brucella suis, Brucella melitensis,
or Brucella abortus
Geographic Distribution: Worldwide, but infections are most
often reported from the southwestern United States, Cen-
tral and South America, China, and Japan
Major Clinical Signs: No signs or infertility, abortion, non-
painful scrotal enlargement and dermatitis, testicular
atrophy, lymphadenopathy, splenomegaly, signs related
to discospondylitis (spinal pain, pelvic limb paresis, or
paralysis), uveitis, chorioretinitis, rarely osteomyelitis and
meningoencephalitis.
Major Mode of Transmission: Direct exposure to contami-
nated body fluids (oral, nasal, conjunctival, venereal)
Differential Diagnoses: Bartonellosis, mycobacterial infec-
tions, chronic vector-borne diseases associated with
splenomegaly such as ehrlichiosis and babesiosis, coxiel-
losis, other bacterial and fungal causes of discospondyli-
tis, intervertebral disc disease, hemic neoplasia (such as
lymphoma or multiple myeloma). Additional differential
diagnoses for dogs with ophthalmitis include deep myco-
ses, rickettsial diseases, leptospirosis, and toxoplasmosis.
Human Health Significance: B. canis can be transmitted to
humans and most often causes mild, influenza-like illness.
Etiology and Epidemiology
Canine brucellosis is caused by Brucella canis, a small, gram-
negative, non–spore-forming aerobic coccobacillus. Brucella
abortus, Brucella melitensis, and Brucella suis occasionally
cause canine infections but are comparatively rare.2,3 B. canis
infections are zoonotic, but other species of Brucella present a
greater risk to human health.4,5
Brucellosis is the primary contagious infectious venereal dis-
ease of concern in canine reproduction. Brucella canis causes
reproductive failure in both male and female dogs. Screening for
B. canis infection is an important part of the pre-breeding evalu-
ation of any dog and should be included in the initial diagnos-
tics in any case of canine abortion, orchitis, epididymitis, and
apparent infertility in bitches or dogs. Because the incidence of
canine brucellosis is low in many geographic locations, breeder
compliance with regular screening can wane, which makes con-
tinued veterinarian vigilance important. Notably, neutered and
maiden or virgin dogs can also be infected. B. canis can also
512
cause systemic disease (such as discospondylitis) in dogs not
used for reproduction. Dogs with B. canis discospondylitis are
typically medium- to large-breed dogs, with body weights that
range from 14 to 64 kg; dogs have ranged in age from 1 to 8
years, with a median of 4 years in one study.6-9 Of 22 dogs with
B. canis discospondylitis known to the authors, 18 (82%) were
intact male dogs, 3 (14%) were female spayed, and 1 (4%) were
male neutered.6,8-10 Three dogs described in the literature with
B. canis ophthalmitis did not fit this signalment; one dog was a
Pomeranian and none were intact males.11
Transmission of B. canis occurs through direct exposure to
body fluids that contain sufficient numbers of bacteria to cause
infection (2 × 106 CFU), most often semen, lochia, aborted
fetuses/placentas, milk, and/or urine. Transmission is therefore
primarily oral, nasal, or conjunctival and secondarily venereal
(i.e., through the mucous membranes). Oronasal and conjuncti-
val exposure follows the ingestion or aerosolization of infectious
materials. The aerosol route is especially important in crowded
kennel conditions. Transplacental transmission and direct cuta-
neous inoculation can also occur. Although fomite-associated
transmission can occur under appropriate circumstances,
B. canis is short-lived outside the host and is readily inactivated
by common disinfectants, such as quaternary ammonium com-
pounds, 1% sodium hypochlorite, 70% ethanol, iodine/alcohol
solutions, and glutaraldehyde.12
Members of the Canidae family are the natural hosts of
B. canis. Canine brucellosis occurs most commonly as outbreaks
in large commercial kennels, and less commonly in privately
owned dogs. Although the geographic distribution of canine
brucellosis is not fully understood, outbreaks of canine brucel-
losis have geographic orientation, with increased incidence seen
in the southernmost United States and commonly in Mexico,
Central and South America, the People’s Republic of China,
and Japan. There are sporadic reports of disease in Canada and
throughout Europe.7,13-17 Australia and New Zealand appear
to be free of the disease. However, because infection with B.
canis is chronic and subclinical, the organism can readily be
imported into regions where the prevalence of disease is low
through the movement of infected dogs. The increased practice
and success of canine semen processing for exportation (chill-
ing and cryopreservation) for the purpose of artificial insemi-
nation now makes canine brucellosis a concern worldwide, as
direct mucosal contact among dogs is no longer necessary for
transmission.
Clinical Features
Signs and Their Pathogenesis
Brucella organisms attach to and penetrate mucous membranes;
the severity of disease is proportional to the infectious dose. After

FIGURE 53-1  Midterm abortion from a Brucella canis affected bitch diagnosed by
AGID. Note the variation in fetal development indicating that death occurred at different
stages. (Courtesy Patricia Olson, DVM, PhD, DACT.)
replication in regional lymph nodes, bacteremia occurs within
7 to 30 days after exposure with subsequent dissemination to
reticuloendothelial cells, the prostate, uterus, and placenta.
B. canis has a predilection for steroid-dependent (reproductive)
tissues. The organism survives facultatively within monocytes
and macrophages of the reticuloendothelial system. Brucella
organisms can survive and multiply in reticuloendothelial cells
because they inhibit the bactericidal myeloperoxidase-peroxide-
halide system through the release of 5′-guanosine and adenine.
Early in infection, polymorphonuclear cells and macrophages
kill intracellular B. canis relatively ineffectually.18
Brucella organisms can be found in the rough endoplasmic
reticulum of placental trophoblastic giant cells in an infected
bitch’s gravid uterus. Severe necrotizing placentitis with infarc-
tion of the labyrinth region, coagulation necrosis of the chorionic
villi, and necrotizing arteritis result in fetal death. The organism
can be found in the gastric contents of aborted fetuses. Necro-
tizing vasculitis and granulomatous inflammation results in epi-
didymal and subsequent testicular and prostatic pathology.3,19
Brucellosis in dogs is associated with a high morbidity but
low mortality. The clinical systemic signs are often subtle and
include suboptimal athletic performance, lumbar pain, lameness,
weight loss, and lethargy. The primary clinical sign of canine
brucellosis in the breeding bitch is pregnancy loss, which can
occur early (day 20) in gestation resulting in fetal resorption, or
more commonly (75% of dogs) later in gestation (generally 4 to
59 days), which results in abortion (Figure 53-1). Bitches with
pregnancy loss early in gestation can appear to be infertile (fail
to conceive) unless early ultrasonographic pregnancy evaluation
is performed to identify pregnancy. Nongravid bitches can show
no signs or can show regional lymphadenopathy (pharyngeal if
orally acquired, inguinal and pelvic if venereally acquired).
The primary acute clinical signs of canine brucellosis in the
male dog involve the portions of the reproductive tract that
participate in the maturation, transport, and storage of sper-
matozoa. Epididymitis is common, with associated orchitis and
scrotal dermatitis, and resultant deterioration of semen qual-
ity and fertility. Chronically, testicular atrophy and infertility
can occur. The organism can be found in the prostate gland
CHAPTER 53  Canine Brucellosis 513
FIGURE 53-2  Brucella discospondylitis at the T7-T8 junction with ventral spondylosis
and ­collapse of the intervertebral disc space. (Courtesy Anna Park, DVM, and James Lavely,
DVM, DACVIM [Neurology].)
and urethra and is shed intermittently in the urine. Anti-sperm
antibodies develop in association with brucellosis-induced
epididymal granulomas and further contribute to infertility.
Pyospermia develops 3 to 4 months postinfection. Chronic
infections in either sex can result in uveitis or endophthalmitis,
lymphadenitis, splenomegaly, or discospondylitis; occasionally,
dermatitis and meningoencephalitis have been reported (Figure
53-2).3,8,11,20 Owners of dogs with discospondylitis most often
describe episodic neck pain or lameness, which can occasion-
ally be accompanied by lethargy or decreased activity. Bacte-
remia can persist for years, and subclinically infected dogs can
remain infectious for long intervals. Infection of the bone that
surrounded a total hip replacement was described in two dogs
9 and 12 months after surgery.21
Large numbers of organisms are shed in the vulvar discharge
of bitches 4 to 6 weeks postabortion. The highest concentration
of organisms is shed in the semen of infected dogs 2 to 3 months
postinfection, with lesser amounts for years. Urine can serve as
a contaminated vehicle because of the proximity of the urinary
and genital tracts in the dog, with shedding present for months
to years, and is more prevalent in males. Organisms can also be
shed in milk.22-24
Spontaneous apparent recovery can occur 1 to 5 years
postinfection, but true clearance of the organism is difficult
to document. Bitches that have been treated with antimicro-
bial drugs can produce normal litters after multiple abortions,
but can remain infectious to their offspring. Dogs may remain
infertile because of irreparable damage to the spermatogenic
apparatus.3,23
Physical Examination Findings
For dogs with clinical B. canis infections, fever is rarely pres-
ent. Affected male dogs may have an enlarged but nonpain-
ful scrotum and secondary scrotal pyoderma, which results
from excessive licking. Enlargement of the epididymis is often
present. Some chronically infected male dogs have small, soft
testicles due to testicular atrophy. Other dogs have general-
ized peripheral lymphadenomegaly, and splenomegaly may be
detected on abdominal palpation. The most common finding
in dogs with discospondylitis is spinal pain.8,9 Other findings
514 SECTION 2  Bacterial Diseases

TABLE 53-1
Diagnostic Assays Available for Brucella Infection in Dogs
Assay Specimen Type Target Performance
Culture Whole blood, urine, disc aspirates or Brucella spp. Confirms infection but is insensitive. Antibiotic
bone fragments, tissue specimens treatment can lead to false-negative results.
obtained at necropsy (e.g., spleen,
uterus, prostate), aborted fetuses
and placental tissue, seminal fluid
Histopathology Tissue specimens obtained by biopsy Brucella spp. IHC may have low sensitivity depending on the
with organism or at necropsy organisms tissue examined, but supports disease causa-
detection using tion when positive and properly performed and
IHC interpreted.
Serology, rapid Serum Antibodies to Rapid and inexpensive. False positives are com-
slide agglu- Brucella spp. mon as a result of cross-reactivity with other
tination test bacterial species and require confirmation with
(RSAT), and an alternative test. Positive results do not imply
mercaptoetha- active infection. False-negative results with RSAT
nol RSAT can occur in dogs with discospondylitis. The
sensitivity of the RSAT may be higher than that
of the ME-RSAT.
Serology, tube ag- Serum Antibodies to See RSAT. Usually positive by 2 weeks after infection.
glutination test Brucella spp. Titers ≥ 1:200 tend to correlate with bacteremia.
Serology, agar gel Serum Antibodies to See RSAT. Requires special expertise and is time-
immunodiffu- Brucella spp. consuming to perform. May not be positive until
sion 12 weeks after infection. Cytoplasmic antigen as-
says are more specific for Brucella spp. antigens.
Serology, indirect Serum Antibodies to See RSAT. Requires expertise to perform and inter-
immunofluores- Brucella spp. pret. May have lower specificity and sensitivity
cent antibody when compared with ELISA assays or the tube
(IFA) agglutination test.
Serology, ELISA Serum Antibodies to Has the potential to be sensitive and specific, but
Brucella spp. assays vary in performance. A standard, accepted
assay is not currently available.
PCR Whole blood, splenic or lymph node Brucella spp. Confirms active infection and has the potential to
aspirates, disc aspirates, tissue DNA be more sensitive than culture. Sensitivity and
specimens obtained at necropsy, specificity may vary depending on assay design,
vaginal swabs, seminal fluid, urine, specimen type, and quality assurance practices
placental materials, or fetuses in the laboratory. Results should be interpreted
with caution and in light of the clinical picture
and the results of serologic tests and/or culture.

IHC, immunohistochemistry.

include lameness, delayed placing reactions, ataxia, pelvic limb
hyperreflexia, and, uncommonly, pelvic limb paresis or tetra-
paresis. Meningoencephalitis may result in behavioral changes,
anisocoria, ataxia, circling, or a head tilt. For unknown rea-
sons, ocular abnormalities are often unilateral and include
blepharospasm, conjunctival hyperemia, and evidence of uve-
itis, such as aqueous flare, miosis, iris hyperpigmentation, loss
of the normal contour of the iris surface, hypopyon, posterior
synechiae, and secondary glaucoma.11 Corneal edema, vitre-
ous opacities, swelling and hyperemia of the optic disc, and
evidence of multifocal granulomatous chorioretinitis have also
been described. Ocular abnormalities and discospondylitis are
frequently present in the absence of other systemic signs of ill-
ness. However, some, but not all dogs with discospondylitis
have other, subtle physical examination abnormalities that
suggest brucellosis, such as dermatitis, uveitis, small testicles,
or lymphadenopathy.8
Diagnosis
The diagnosis of canine brucellosis is based on suggestive
clinical signs and the results of serology; culture of blood,
urine, or tissues; histopathology; and/or PCR assays. Because
no single antemortem test has 100% sensitivity, and serologic
assays lack specificity, a combination of diagnostic assays is
often required to make a diagnosis (Table 53-1). When mul-
tiple dogs with infertility problems are present in a household
or kennel, as many dogs as possible should be tested with
serology and blood culture to increase the chance of a reliable
diagnosis.
 CHAPTER 53  Canine Brucellosis 515

A B
FIGURE 53-3  Radiographic images from a 1-year-old female spayed labradoodle with Brucella canis discospondylitis that was evaluated for cervical pain. CSF analysis revealed a CSF
protein concentration of 31 g/dL, with 3 nucleated cells/µL, 89% of which were small mononuclear cells and 11% of which were large mononuclear cells (marginal lymphocytic pleocyto-
sis). Brucella canis was isolated from 5 of 5 blood culture specimens. Urine culture was negative. Of significance, a B. canis rapid slide agglutination test was negative. The dog was treated
successfully with antimicrobial drugs (doxycycline and clavulanic acid–amoxicillin) and lesions resolved radiographically. A, Plain lateral radiograph of the cervical spine. There is endplate
lucency associated with cranial endplate of C3 at the C2-3 articulation. The C2-3 disc space appears narrowed. B, Computed tomography image. Central lysis of the end plates at C2-3 is
present (arrow), compatible with discospondylitis at C2-3.

Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
The results of routine laboratory tests are often within reference
ranges in dogs with brucellosis. The CBC may show leukocyto-
sis due to a neutrophilia.11 Degenerate left shifts, monocytosis,
and/or lymphopenia have been uncommonly reported.9,20 The
chemistry panel is often normal, but hyperglobulinemia and
hypoalbuminemia may be present. A lack of abnormalities on
routine laboratory testing or the presence of splenomegaly or
abdominal lymphadenomegaly in dogs with discospondylitis
should raise suspicion for B. canis infection. Urinalysis is gen-
erally within normal limits, even when B. canis bacteriuria is
present.
Brucella. Detached sperm heads, proximal and distal cytoplas-
mic droplets, and acrosomal deformities are the most common
morphologic abnormalities. Sperm head-to-head agglutination
suggests the presence of anti-sperm antibodies. An absence of
sperm in the ejaculate (azoospermia) can also be found.
Diagnostic Imaging
Plain Radiography and Advanced Imaging
Plain radiography may show evidence of either multifocal
or focal discospondylitis (see Figure 53-2; Figure 53-3, A).
Advanced imaging (CT or MRI) may be more sensitive than
plain radiographs for detection of endplate lucency (see Figure
53-3, B; see also Chapter 85).

Cytologic Analysis of Tissue Aspirates Sonographic Findings

Aspirates of the spleen or lymph nodes of dogs with brucellosis
reveal lymphoid reactivity, sometimes with plasmacytosis. Pros-
tatic aspirates in one dog with prostatitis showed granulomatous
inflammation with intracellular gram-negative coccobacilli.7
Cytologic Analysis of Body Fluids
CSF analysis in dogs with Brucella discospondylitis is usually
unremarkable. A dog with meningoencephalitis had neutro-
philic pleocytosis and increased CSF protein concentration.3
Aspirates of ocular fluids (aqueous or vitreous humor) from a
dog with panophthalmitis revealed pyogranulomatous inflam-
mation with extracellular and intracellular small gram-negative
cocci.11
Semen Examination
Diminished sperm counts (oligospermia), poor motility (asthe-
nospermia), and increased morphologic abnormalities (tera-
tospermia) are characteristic of semen in dogs infected with
Abdominal ultrasound examination of dogs with brucellosis
may show no abnormalities, or abdominal lymphadenomegaly
and splenomegaly may be present. Ultrasound of the pros-
tate may reveal prostatic irregularity, hyperechogenicity, and
cavitation.7
Microbiologic Tests
Serologic Diagnosis
There are two reasons to perform serologic tests for brucellosis
in dogs: (1) as part of a routine screening program in breed-
ing kennels, whereby healthy dogs are screened for infection,
and (2) for diagnosis of active infection in sick dogs that are
suspected to have brucellosis because of infertility, reproduc-
tive losses, epididymitis, discospondylitis, osteomyelitis, or
ophthalmitis.
Assays used for screening purposes are usually rapid and
inexpensive, but can suffer from low specificity (a high rate of
false-positive test results) due to strong cross-reactivity between
516 SECTION 2  Bacterial Diseases
surface lipopolysaccharide (LPS) antigens of B. canis and those
of other nonpathogenic infectious agents. Up to 50% to 60% of
dogs can have false-positive test results because of cross-reacting
antibodies to microorganisms such as Bordetella, Pseudomonas,
Escherichia coli, and Moraxella spp. Even when the antibod-
ies detected truly represent anti-Brucella antibodies, the pres-
ence of these antibodies does not indicate active infection with
Brucella because dogs can remain seropositive for months after
they clear the infection. Antibodies to Brucella do not become
detectable until 2 to 12 weeks after infection, so a window of
time exists in which an infected dog eludes serologic diagno-
sis with any assay used. False negatives also occasionally occur
in dogs with chronic sequestered infections. Beyond the first 3
months of infection, the sensitivity of screening assays is gener-
ally believed to be high, but in some scenarios it might be lower
than previously thought.25 Examples of screening assays include
the rapid slide agglutination test (RSAT); the semiquantitative
2-mercaptoethanol modified RSAT (ME-RSAT); the semiquan-
titative tube agglutination test (TAT); agar gel immunodiffusion
(AGID) assays; and indirect IFA or ELISA assays. Veterinar-
ians need to understand the limitations of the assays available
to them through local veterinary diagnostic laboratories. The
RSAT uses Brucella ovis as the antigen, whereas the ME-RSAT
uses B. canis antigen, which may improve specificity. However,
in one study, the sensitivity of the ME-RSAT was only 32%,
when compared with 71% for the RSAT, so the RSAT may
be preferable for screening.25 In the TAT, a titer of more than
1:200 correlates well with positive blood cultures, but lower
titers are more difficult to interpret. The performance of AGID
assays requires trained laboratory personnel and special media.
Two types of AGID assay are available: an assay that uses cell
wall antigens as the substrate (cell wall AGID assay) and an
assay that uses cytoplasmic antigens of Brucella as the substrate
(cytoplasmic AGID assay). The cell wall assay has from the same
low specificity as other assays that detect antibodies to Brucella
LPS, and the sensitivity may be lower than that of the RSAT.25
In contrast, the cytoplasmic assay is specific for antibodies to
Brucella, so, when available, it is a useful confirmatory assay for
dogs that test positive with other rapid serologic assays. Indi-
rect IFA has lower sensitivity than many other serologic assays.
A number of ELISA assays have been developed to overcome
difficulties that relate to the low specificity of serologic assays
for B. canis and the limited availability of other assays. The
performance of these ELISA assays has varied, but sensitive and
specific ELISA assays have been developed that may prove use-
ful for rapid serologic testing in the future.26
In practice, for screening purposes, healthy bitches are tested
at least 1 month before a planned breeding or introduction to a
breeding kennel with a serologic assay of low specificity such as
the RSAT because these assays are rapid, readily available, and
inexpensive. Healthy, fertile stud dogs should be tested annually
with the RSAT. If the test result is negative, in cases of subfertil-
ity or if recent exposure is suspected, a second test is performed
a month later, to assess for seroconversion (which would be
expected if the dog was early in the course of infection when
first tested). If the test result is positive, a confirmatory assay
should be performed. This could include the TAT or a more spe-
cific assay, such as the cytoplasmic AGID assay, a specific ELISA
assay, blood culture, or a reliable PCR assay, although blood
culture can lack sensitivity.13,15 Usually the assay(s) selected is
based on test availability and financial resources of the owner.
Positive serologic test results for dogs with discospondylitis or
other disease manifestations suggestive of brucellosis should be
confirmed using culture or a reliable PCR assay whenever pos-
sible. Although false-negative serologic test results are uncom-
mon in dogs with discospondylitis, negative serologic test
results in dogs with discospondylitis do not rule out infection
with Brucella spp., and attempts should also be made to detect
the organism with culture or PCR assays. Of various dogs with
B. canis discospondylitis or ophthalmitis, 15 of 18 were positive
with the RSAT, 16 of 16 were positive with the TAT, and 6 of
7 were positive with AGID assays.6,8-11 The 3 dogs that were
negative with the RSAT had discospondylitis. One was positive
by blood culture, and the other 2 dogs tested positive with the
TAT at a later date.8,10
Isolation and Identification
Despite improvements in serologic diagnostic methods, con-
firmatory blood cultures are still indicated when brucellosis is
suspected. B. canis is readily isolated from the blood of bacte-
remic individuals for several months after infection. Specimens
for blood culture should be collected as detailed in Chapter
3. A positive B. canis culture has been advocated as the best
diagnostic test in the first 2 months of the disease; however,
dogs can become abacteremic 27 to 64 months after infection
but remain infected. Occasionally B. canis can be isolated from
urine, tissue specimens such as aborted fetuses or placental
material, necropsy specimens (especially spleen, prostate, and
uterus), vaginal swabs, or disc aspirates. Of dogs with Brucella
discospondylitis or ophthalmitis, 6 of 15 dogs tested positive by
blood culture, 3 of 13 by urine culture, and 4 of 6 by culture of
disc material or bone chips from the intervertebral disc space
obtained at surgery.6,8-10
B. canis is an aerobe, but unlike other Brucella species, added
CO2 (5% to 10%) can be inhibitory. Multiplication is slow at
the optimum temperature of 37°C, and enriched medium (tryp-
tose or trypticase soy media) is occasionally needed to support
adequate growth. Brucella colonies become visible in 2 to 3 days.
Growth of Brucella can be identified on the basis of rough (as
opposed to smooth) colony morphology, staining characteris-
tics, and slide agglutination with anti-Brucella serum. Identifica-
tion to the species level is best done in a specialized laboratory.
Differentiation between B. canis and B. suis can be difficult.13 In
the United States, the New York State Diagnostic Laboratory at
Cornell University, the Tifton Veterinary Diagnostic and Inves-
tigational Laboratory in Georgia, and the University of Florida
are recognized to be reliable for definitive testing made neces-
sary by positive B. canis screening tests in dogs intended for
breeding, or for clinically affected dogs undergoing evaluation
for infertility or abortion, or when outbreaks are being man-
aged.3,27 Susceptibility testing can be performed, but in vitro test
results may not correlate well to the in vivo treatment response.
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays have been used to successfully identify B. canis in
semen from dogs with negative culture results, which suggests
that this may ultimately be the most sensitive method of testing as
part of the male dog pre-breeding evaluation, especially in cases
where management of an outbreak is the concern.28 PCR assays
were also used to detect B. canis in the aqueous humor of dogs
with endophthalmitis11 and in lymph node aspirates.29 Assays
have been designed that differentiate between the Brucella species
present.30 A real-time PCR assay was used to detect B. canis in tis-
sue specimens, urine, buffy coat, and intervertebral disc material

of a dog with discospondylitis when culture of intervertebral disc
material, blood, and urine was negative.7 Some veterinary diag-
nostic laboratories in the United States now offer real-time PCR
assays for detection of B. canis on a commercial basis.
Pathologic Findings
The main pathologic findings of B. canis infections consist of
lymphoplasmacytic to lymphohistiocytic follicular hyperplasia
in lymphoid tissues, lymphohistiocytic to neutrophilic endo-
phthalmitis, and mild lymphohistiocytic meningitis. Occasion-
ally, mild lymphoid and histiocytic infiltrates are present in other
tissues, such as the liver and lung.15 A mixed lymphohistiocytic
inflammatory response is also present in the uterus (endometri-
tis) of females and the testicles, epididymis and spermatic cord,
and prostate of male dogs. Prostatic and testicular fibrosis may
be found. Placental trophoblasts can contain large numbers of
gram-negative bacteria, and coagulative necrosis may be pres-
ent. In male dogs, lesions are more likely to be restricted to the
reproductive system. Immunohistochemistry has been used to
confirm the presence of Brucella spp. in placental tissues.13
Treatment and Prognosis
In certain jurisdictions, brucellosis is a reportable disease
in dogs or in humans. Infected dogs and bitches should be
removed from breeding programs and quarantined. Eradication
of the disease in kennel situations has not been successful with-
out removal (culling) of all current or historically infected dogs.
Because of the zoonotic potential of the disease and difficulty in
eradicating the infection, euthanasia of affected dogs has been
recommended. Infection in household or small hobby kennel
dogs often results in client requests for alternatives to euthana-
sia. Neutering decreases the amount of organism shed in semen
and uterine discharge, but does not eradicate infection. Urine
shedding can persist, and the organism can be found in internal
organs and the bloodstream.
Antibiotic therapy has historically not been rewarding, likely
because the organism is intracellular and bacteremia is peri-
odic. Antimicrobial drug treatment can reduce antibody titers
without clearing infection. Relapses are common. Combination
therapy should be used whenever possible, preferably with tet-
racyclines (high-dose doxycycline or minocycline) for at least 1
to 2 months and an aminoglycoside (streptomycin or gentami-
cin) for the first 1 to 2 weeks of treatment (Table 53-2). Some
also advocate use of two 1-week courses of aminoglycoside
TABLE 53-2
Medications That Could Be Used to Treat Brucella spp. Infections
in Dogs
Interval
Drug Dose (mg/kg) Route (hours)
Doxycycline* 10-15 PO 12
Streptomycin 20 IM 24
Gentamicin sulfate 9-14 IV, IM, SC 24
Rifampin 5 PO 12
Enrofloxacin 5-10 PO 24
*Use in combination with one of the other drugs listed. Combinations
of drugs are preferred for treatment where possible.
CHAPTER 53  Canine Brucellosis 517
treatment spaced 1 month apart, or treatment with streptomy-
cin every other week for 8 weeks.11 Combination therapy with
tetracyclines and streptomycin is thought to be the most suc-
cessful treatment, but unavailability of streptomycin, nephro-
toxicity, parenteral therapy requirements, and expense may be
problematic.31-33 In addition, aminoglycosides do not provide
adequate ocular or CNS penetration for dogs with ophthalmitis
or meningitis. A combination of doxycycline and rifampin has
been used to treat human brucellosis but may not be well tol-
erated by dogs because of gastrointestinal adverse effects. The
use of three or four drugs in combination (e.g., streptomycin,
enrofloxacin, doxycycline with or without rifampin) was effi-
cacious for treatment of ophthalmitis in dogs.11 Topical 1%
prednisolone acetate and atropine ointment may be required for
management of uveitis.11
One study reported a possible benefit of therapy with enro-
floxacin (5 mg/kg q12h PO for 4 weeks, often for multiple
courses) in a small group of infected dogs and bitches. Enro-
floxacin did not completely eliminate B. canis, but it maintained
fertility and avoided the recurrence of abortions, transmis-
sion of the disease to subsequently whelped puppies, and dis-
semination of microorganisms during parturition. Ultimately,
however, most treated individuals remained culture positive.34
Combinations of enrofloxacin and doxycycline may be a more
effective alternative for dogs unable to tolerate aminoglycoside
or rifampin treatment and for dogs that have ocular or CNS
involvement, but studies are lacking.
Vaccination and Immunity
Serum agglutinating antibodies do not protect against infec-
tion in the dog, and immunity likely depends on cell-mediated
immunity. Presently, the development of a vaccine is consid-
ered undesirable because the Brucella vaccines evaluated have
offered only moderate protection, and immunized dogs develop
antibodies that confound serodiagnosis.
Prevention
Prevention of infection and elimination of infected dogs should
be the principal control strategy in kennels. Prevention requires
at least yearly testing of all breeding stock and the testing of
all dogs to be introduced into a kennel. Ideally, two negative
screening tests performed at least a month apart should occur
before a dog or bitch is introduced into a breeding facility. Con-
firmed positive dogs should be isolated (euthanasia is advised by
many authors), neutered, treated, and tested monthly (AGID,
culture, or reliable PCR assay) until two consecutive negative
tests occur, although occult infection can still be present.
Private breeders should require serologic screening of all
bitches offered for breeding and confirmatory negative test
results if positive results occur during screening before they
accept a bitch into their kennel. Stud dogs should be screened at
least annually. Because of the potential for nonvenereal trans-
mission, screening of maiden dogs and bitches before breeding
is also recommended.35,36
Public Health Aspects
Humans can become infected with B. canis, although they
are relatively resistant to infection and other Brucella species
account for the majority of human disease. Approximately 40
518 SECTION 2  Bacterial Diseases
cases of human infection have been reported worldwide; how-
ever, the actual number is unknown. Serologic assays used for
diagnosis of brucellosis in humans often do not detect antibod-
ies to B. canis, so the disease may be overlooked. Transmis-
sion to humans most commonly occurs through contact with an
infected dog’s semen, vulvar discharge from an infected bitch, or
aborted fetuses or placentas, or through direct, accidental labo-
ratory exposure.8 Thus, disease is more likely to occur in people
who are in contact with breeding dogs, but disease occasionally
occurs in the owners of pet dogs with discospondylitis.10 Immu-
nocompromised humans, children, and those who are pregnant
may be at greater risk for development of disease. Infection of
humans with B. canis typically results in nonspecific, persistent
clinical signs of fever, muscle aches, night sweats, and general
malaise. Rarely, granulomatous hepatitis, hepatosplenomeg-
aly, osteomyelitis, pulmonary disease, and endocarditis have
been reported. In one case report, disease occurred in an HIV-
infected dachshund dog breeder who reported infertility in one
of the dogs, which had been tested by a veterinarian and was
seropositive with a screening assay. Confirmatory testing with
CASE EXAMPLE
Signalment: “Sadie”, a 6-year-old female spayed Border
collie mix from northern California
History: Sadie was brought to her local veterinarian for
listlessness and reluctance to jump noted during the
previous 48 hours. She had been adopted from a local
humane shelter as an ovariohysterectomized 11-month-
old. She lived in a rural agricultural area in northern
California and was the only dog in the household.
Physical Examination: Sadie’s vital signs were within
normal limits, mucous membrane color was pink,
and she was quiet and alert on physical examination.
Body weight was 25 kg. Mild dental tartar was evident.
Thoracic auscultation and abdominal palpation were
normal, pulses were synchronous, and no peripheral
lymphadenomegaly was evident. Palpable dorsal
thoracolumbar pain was demonstrated. She was reluctant
to rise but was ambulatory. The neurologic examination
including a fundoscopic examination was normal.
Laboratory Findings: A complete blood count, chemistry
panel, and urinalysis were performed. Mild neutropenia
(2958 cells/µL; reference range, 3000-11,500 cells/µL) was
found, and the chemistries and urinalysis were normal
with no proteinuria.
Imaging Findings: Radiographs of the spine revealed
evidence of disc space collapse, spondylosis, and
discospondylitis at the T7-T8 disc space (see Figure 53-2).
Microbiologic Testing: A SNAP 4DX test (IDEXX
Laboratories) for antigen of Dirofilaria immitis and
antibodies to Ehrlichia canis, Anaplasma phagocytophilum,
and Borrelia burgdorferi was negative. Aerobic bacterial
urine culture was negative. Serum was submitted for a
AGID performed at a university veterinary diagnostic labora-
tory yielded a negative result, so brucellosis was not thought
to be present. However, subsequent blood culture of all of the
dogs in the household was positive in one other dog.37 Affected
humans are typically treated successfully with combinations of
doxycycline and either aminoglycosides (for the first week) or
rifampin, but monotherapy has been associated with unaccept-
able rates of relapse.
Prevention of human disease involves eradication of the dis-
ease in dog kennels; precautions such as the wearing of gloves
and protective clothing when in contact with aborted mate-
rial; and hand washing after handling dogs and before eating.
Although it is preferable that immunocompromised individu-
als do not participate in dog breeding activities, if necessary,
these individuals should take special care if they interact with
breeding dogs. Contact with dogs with reproductive problems
or their secretions should be avoided whenever possible, and if
aborted material must be handled, gloves, gowns, and face pro-
tection should be worn, all protective wear should be disposed
of properly, and hands should be washed thoroughly.
Brucella canis IFA, which was positive at a titer of 1:200. A
B. canis AGID was submitted and was also positive.
Treatment: Sadie was treated with tramadol (1-2 mg/
kg PO q8h) and carprofen (3 mg/kg PO q12h). Once
the serologic test results were available, a tentative
diagnosis of B. canis discospondylitis was made, and
she was treated with enrofloxacin (5.5 mg/kg PO q12h).
Clinical improvement was reported in 2 weeks, and
repeat radiographs were performed in 1 and 5 months.
Equivocal change was reported in the appearance of T7-
T8 at 1 month, but the 5-month radiograph showed no
progression, suggesting the lesion was no longer active.
The owners were advised of the zoonotic potential of this
disease and the potential hazard of transmission to other
dogs. The potential need for lifelong antibiotic therapy
with risk of relapse was also discussed.
Comments: Dogs with Brucella discospondylitis frequently
are brought to their veterinarian for clinical signs of spinal
pain or lameness that resemble common musculoskeletal
injuries such as intervertebral disc disease or rupture of
the cranial cruciate ligament. Although not typical, this
case illustrates the fact that B. canis disease can occur in
female spayed, nonbreeding dogs and often occurs in the
absence of fever or significant abnormalities on routine
laboratory testing. A diagnosis of Brucella discospondylitis
was strongly suspected based on positive serologic
test results with multiple assays. Blood culture was not
performed, but urine culture was negative. Monotherapy
with fluoroquinolones has been associated with high
rates of relapse in human patients once antimicrobial
drug treatment has been discontinued, so it is generally
not recommended. Nevertheless, it can occasionally be
successful.

SUGGESTED READINGS
Hollett RB. Update on canine brucellosis. Theriogenology. 2010;1:
287-295.
Lawaczeck E, Toporek J, Cwikla J, et  al. Brucella canis in a HIV-
infected patient. Zoonoses Public Health. 2011;58:150-152.
Wanke MM. Canine brucellosis. Anim Reprod Sci. 2004;82-83:195-
207.
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1. Carmichael LE. Abortion in 200 beagles. J Am Vet Med Assoc.
1966;149:1126.
2. Barr SC, Eilts BE, Roy AF, et al. Brucella suis biotype 1 infection in
a dog. J Am Vet Med Assoc. 1986;189:686-687.
3. Greene CE, Carmichael LE. Canine brucellosis. In: Greene CE, ed.
Infectious Diseases of the Dog and Cat. 4th ed. St. Louis, MO:
Elsevier Saunders; 2012:398-411.
4. Blankenship RM, Sanford JP. Brucella canis. A cause of undulant
fever. Am J Med. 1975;59:424-426.
5. Lucero NE, Escobar GI, Ayala SM, et al. Diagnosis of human brucel-
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6. Anderson GI, Binnington AG. Discospondylitis and orchitis associ-
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7. Corrente M, Franchini D, Decaro N, et  al. Detection of Brucella
canis in a dog in Italy. New Microbiol. 2010;33:337-341.
8. Kerwin SC, Lewis DD, Hribernik TN, et al. Diskospondylitis asso-
ciated with Brucella canis infection in dogs: 14 cases (1980-1991).
J Am Vet Med Assoc. 1992;201:1253-1257.
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in three dogs infected with Brucella canis. J Am Vet Med Assoc.
1974;165:451-455.
10. Sykes JE. Unpublished observations, 2012.
11. Ledbetter EC, Landry MP, Stokol T, et  al. Brucella canis endo-
phthalmitis in 3 dogs: clinical features, diagnosis, and treatment.
Vet Ophthalmol. 2009;12:183-191.
12. Carmichael LE, Joubert JC. Transmission of Brucella canis by con-
tact exposure. Cornell Vet. 1988;78:63-73.
13. Brennan SJ, Ngeleka M, Philibert HM, et al. Canine brucellosis in
a Saskatchewan kennel. Can Vet J. 2008;49:703-708.
14. Dunne J, Sehgal K, McMillan A, et al. Canine brucellosis in a dog
imported into the UK. Vet Rec. 2002;151:247.
15. Gyuranecz M, Szeredi L, Ronai Z, et  al. Detection of Brucella
canis–induced reproductive diseases in a kennel. J Vet Diagn Invest.
2011;23:143-147.
16. Nockler K, Kutzer P, Reif S, et  al. Canine brucellosis—a case
report. Berl Munch Tierarztl Wochenschr. 2003;116:368-372.
17. Strom Holst B, Lofqvist K, Ernholm L, et al. The first case of Bru-
cella canis in Sweden: background, case report and recommen-
dations from a Northern European perspective. Acta Vet Scand.
2012;54:18.
18. Shin SJ, Carmichael LE. Canine brucellosis caused by Brucella
canis. In: Carmichael LE, ed. Recent Advances in Canine Infectious
Diseases. Ithaca, NY: International Veterinary Information Service;
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19. Moore JA, Kakuk TJ. Male dogs naturally infected with Brucella
canis. J Am Vet Med Assoc. 1969;155:1352-1358.
20. Dawkins BG, Machotka SV, Suchmann D, et al. Pyogranulomatous
dermatitis associated with Brucella canis infection in a dog. J Am
Vet Med Assoc. 1982;181:1432-1433.
21. Smeak DD, Olmstead ML, Hohn RB. Brucella canis osteomyelitis
in two dogs with total hip replacements. J Am Vet Med Assoc.
1987;191:986-990.
22. Carmichael LE, Kenney RM. Canine abortion caused by Brucella
canis. J Am Vet Med Assoc. 1968;152:605-616.
23. Johnson CA, Walker RD. Clinical signs and diagnosis of Brucella
canis infection. Compend Cont Educ Pract Vet. 1992;14:763-772.
24. Schoeb TR, Morton R. Scrotal and testicular changes in canine bru-
cellosis: a case report. J Am Vet Med Assoc. 1978;172:598-600.
25. Keid LB, Soares RM, Vasconcellos SA, et al. Comparison of agar
gel immunodiffusion test, rapid slide agglutination test, microbio-
logical culture, and PCR for the diagnosis of canine brucellosis. Res
Vet Sci. 2009;86:22-26.
26. de Oliveira MZ, Vale V, Keid L, et  al. Validation of an ELISA
method for the serological diagnosis of canine brucellosis due to
Brucella canis. Res Vet Sci. 2011;90:425-431.
27. Wooley RE, Hitchcock PL, Blue JL, et  al. Isolation of Brucella
canis from a dog seronegative for brucellosis. J Am Vet Med Assoc.
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28. Keid LB, Soares RM, Vasconcellos SA, et al. A polymerase chain
reaction for the detection of Brucella canis in semen of naturally
infected dogs. Theriogenology. 2007;67:1203-1210.
29. Aras Z, Ucan US. Detection of Brucella canis from inguinal
lymph nodes of naturally infected dogs by PCR. Theriogenology.
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for differentiation of Brucella species. Appl Environ Microbiol.
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31. Jennings PB, Crumrine MH, Lewis Jr GE, et al. The effect of a two-
stage antibiotic regimen on dogs infected with Brucella canis. J Am
Vet Med Assoc. 1974;164:513-514.
32. Johnson CA, Bennett M, Jensen RK, et al. Effect of combined anti-
biotic therapy on fertility in brood bitches infected with Brucella
canis. J Am Vet Med Assoc. 1982;180:1330-1333.
33. Nicoletti P. Further studies on the use of antibiotics in canine bru-
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34. Wanke MM, Delpino MV, Baldi PC. Use of enrofloxacin in the
treatment of canine brucellosis in a dog kennel (clinical trial). The-
riogenology. 2006;66:1573-1578.
35. Hollett RB. Canine brucellosis: outbreaks and compliance. Therio-
genology. 2006;66:575-587.
36. Jones RL, Emerson JK. Canine brucellosis in a commercial breed-
ing kennel. J Am Vet Med Assoc. 1984;184:834-835.
37. Lawaczeck E, Toporek J, Cwikla J, et al. Brucella canis in a HIV-
infected patient. Zoonoses Public Health. 2011;58:150-152.
CHAPTER 54
Tetanus and Botulism
Jane E. Sykes
Overview of Tetanus and Botulism
First Described: The etiology of tetanus was identified in the late
1800s in Germany (Nicolaier).1 C. botulinum and its toxin were
first described in Belgium in 1897 (van Ermengem).2 Before
that, descriptions of both diseases had been recorded; teta-
nus was described by Hippocrates more than 2000 years ago.
Causes: Toxins of Clostridium botulinum (botulism) or Clos-
tridium tetani (tetanus)
Geographic Distribution: Worldwide
Major Mode of Transmission: Ingestion of preformed toxin
(botulism) or cutaneous inoculation of spores into a
wound (tetanus)
Major Clinical Signs: Spastic paralysis (tetanus) or flaccid
paralysis (botulism)
Differential Diagnoses: Differential diagnoses for suspected
botulism include polyradiculoneuritis, paralytic rabies, tick
paralysis, coral snake poisoning, myasthenia gravis, and
ionophore toxicity. For tetanus they include strychnine toxi-
cosis, hypocalcemia, metaldehyde toxicity, and extraocular
myositis
Human Health Significance: Botulism and tetanus are not
directly transmissible from animals to humans, but care
should be taken when handling specimens from animals
with botulism.
Etiology and Epidemiology
Tetanus and botulism are neurologic diseases caused by the
toxins of Clostridium tetani and Clostridium botulinum that
occur worldwide. These organisms are gram-positive, sluggishly
motile, anaerobic spore-forming bacilli. The spores of C. tetani
and C. botulinum are extremely resistant in the environment
and resist boiling and disinfection with alcohol or formalin.
Botulism
In dogs and especially cats, botulism is rare and usually follows
ingestion of preformed neurotoxin within carrion, especially
waterfowl or poultry carrion. Disease in dogs can coincide with
outbreaks of botulism in waterfowl. Botulism outbreaks in dogs
have been reported in hunting foxhounds.3,4 Most affected dogs
are medium- to large-breed, intact male or intact female dogs
that vary in age. The only report of botulism in cats was an out-
break of disease that occurred after several cats were fed pelican
carrion.5 The relative resistance of dogs and cats to botulism has
520
been hypothesized to reflect an adaptive response to a carnivo-
rous lifestyle. C. botulinum produces seven related toxins: A, B,
C, D, E, F, and G. Most human botulism is caused by types A,
B, and E.6 Botulism in dogs and cats almost always results from
ingestion of toxin C,3,5,7-9 with the exception of a few dogs in
the Senegal that had type D intoxication.10
Tetanus
C. tetani is widespread in the soil and feces of animals. It pro-
duces two toxins, tetanospasmin and tetanolysin. Tetanolysin
does not appear to have clinical significance. Humans and horses
are most susceptible to tetanus, and so widespread vaccination
against the disease is performed in these species. Disease occurs
occasionally in dogs and is rare in cats. The development of
tetanus in dogs and cats usually follows introduction of spores
into a penetrating wound, but more than one third of affected
dogs have no known wound history (cryptogenic tetanus).11
Traumatic wounds of the feet, claws, or head; draining tracts
associated with grass awn migration; animal bite wounds; tick
bite wounds; bleeding claw trim wounds; wounds that result
from chronic protozoal or fungal infections of the skin; teething
wounds in dogs; and surgical wounds (which include spay and
neuter operations) have all been implicated.11-14 Although dogs
of any age, breed, or sex develop tetanus, disease occurs most
often in young, large-breed, active, intact male dogs.11,13,14 Dis-
ease is seen in puppies as young as 8 weeks of age; some studies
describe a significant proportion of affected dogs as less than
1 year of age. Disease is also more severe in young dogs.11 Cats
are often young cats with outdoor access, but cats of any age
have been affected.15
Clinical Features
Signs and Their Pathogenesis
The toxins of C. tetani and C. botulinum have similar structures
and mechanisms of action, even though they cause diseases that
contrast sharply with one another. Both toxins are among the
most potent toxins known and act at femtomolar concentra-
tions. They consist of two polypeptide chains, a heavy (H) chain
and a light (L) chain, joined by a disulfide bond (Figure 54-1).
The heavy chain attaches to presynaptic nerve terminal mem-
brane receptors, although the precise receptors have not been
identified because of the extremely low concentrations at which
these toxins act. Variation in receptor binding affinities may
explain differences in species susceptibility to clostridial neu-
rotoxins. The toxin is then internalized via receptor-mediated
endocytosis into the presynaptic nerve terminal, where it has a
local impact on nerve cell function (botulinum toxin) and also
travels by retrograde axonal transport to the nerve cell body

FIGURE 54-1  Schematic of the structure of Clostridium tetani and Clostridium botu-
linum toxins.
and diffuses into inhibitory interneurons, where it impacts
inhibitory interneuron function (tetanospasmin). The L chain,
a zinc-dependent matrix metalloproteinase, is released from the
vesicle and subsequently cleaves one or more docking proteins.
These proteins are critical for the release of neurotransmitters
from synaptic vesicles into the synaptic cleft and are located
on the membrane of synaptic vesicles (synaptobrevin) or the
presynaptic cell membrane (syntaxin and SNAP-25). Synapto-
brevin binds to the docking proteins located on the presynaptic
membrane. The resulting complex (the SNARE complex) docks
the synaptic vesicle at the proper location for fusion and exo-
cytosis of neurotransmitter molecules into the synaptic space
(Figure 54-2). Tetanospasmin cleaves synaptobrevin, whereas
botulinum neurotoxin C cleaves syntaxin; other botulinum
neurotoxins cleave synaptobrevin or SNAP-25. The process of
recovery involves the sprouting of new presynaptic terminals,
followed by recovery of the original synapse, after which new
terminal outgrowths retract.16
Botulism
C. botulinum spores germinate in anaerobic conditions and pro-
duce toxin. They grow best in temperatures that range from
30°C to 37°C. The toxin is ingested, survives the acid stomach
conditions, and is absorbed into the bloodstream from the small
intestine. It then travels to peripheral cholinergic synapses,
which include the neuromuscular junction and autonomic syn-
apses, and inhibits release of acetylcholine (ACh). This leads to
ascending symmetrical flaccid paralysis, development of mega-
esophagus, and signs of autonomic dysfunction. Clinical signs
usually occur within 12 to 72 hours after ingestion of botulinum
toxin and may be complicated by signs of dietary indiscretion,
although gastrointestinal signs may also reflect the presence of
autonomic dysfunction. Disease progresses rapidly over 1 or 2
days, and then begins to resolve with supportive care. Death can
result from respiratory muscle paralysis or aspiration of intesti-
nal contents. Abnormalities in affected cats were ascending flac-
cid paralysis, lethargy, hypothermia, tachypnea, dehydration,
and urinary incontinence.5
Tetanus
C. tetani spores are introduced into tissues where they germinate,
replicate locally, and produce toxin in anaerobic conditions.
Ingestion is not a route of transmission, because tetanospasmin
is destroyed by gastrointestinal secretions. The toxin is produced
as a single polyprotein that is cleaved extracellularly by a bacte-
rial protease into the L and H chains, which remain connected
by the disulfide bond. It then binds to presynaptic terminals of
CHAPTER 54  Tetanus and Botulism 521
lower motor neurons and is internalized, travels up axons by
retrograde axonal flow, and enters inhibitory interneurons in
the brain and spinal cord, where it interferes with release of
γ-aminobutyric acid (GABA) and glycine inhibitory neurotrans-
mitters (Figure 54-2, B). Toxin can also spread hematogenously
to the central nervous system (CNS) from the wound site. The
result is unhindered excitation of motor neurons, spastic paraly-
sis, and dysfunction of the sympathetic and parasympathomi-
metic nervous systems. Clinical signs occur 3 days to 3 weeks
after a wound in both cats or dogs. Shorter incubation periods
occur when the wound is proximal to the CNS and when a large
amount of toxin is present. Paralysis may initially be localized
to the wound site but can then generalize. Involvement of the
head is common in dogs, and often owners of dogs with tetanus
first notice an unusual appearance to their dog’s eyes, a “swol-
len” face, or evidence of dysphagia.11 Regurgitation or vomit-
ing of food occurs in less than 20% of affected dogs, and a
voice change or dysuria may be noted. Death occurs in severely
affected dogs due to paralysis of respiratory muscles; infectious
complications such as pneumonia; upper airway obstruction;
the development of severe hyperthermia with disseminated
intravascular coagulation (DIC), multiple organ failure, and/or
pulmonary thromboembolism; and cardiac arrhythmias.11,13,14
Hiatal hernia and coxofemoral joint luxations have also been
described as complications of tetanus in dogs.11,14,17 Disease in
cats is usually localized to the limbs, but generalized tetanus
can occur.15,18-21 Many affected cats have an initial history of
disappearance for days to a week or more, after which lameness
is noted.
Physical and Neurologic Examination Findings
Dogs and cats with botulism or tetanus are usually alert, afe-
brile, and in good body condition, but lethargy and anorexia
occur in some animals. Hyperthermia can occur with severe
tetanus.
Botulism
Physical examination findings in dogs with botulism depend
on the severity of intoxication. Fever is absent unless aspi-
ration pneumonia has developed. Mildly affected dogs show
only ataxia or pelvic limb paresis, whereas dogs with severe
intoxication are quadriplegic with absent voluntary motor
activity and are unable to rise from lateral recumbency. The
ability to wag the tail is often retained.3,8,9 Neurologic exami-
nation can reveal decreased cranial nerve function, especially
facial nerve function and a decreased gag reflex, sometimes
accompanied by ptyalism.3,7,9,22 A weak blink reflex can result
in corneal ulceration. A mucopurulent ocular discharge may
be present. Mydriasis with sluggish pupillary light reflexes
has been described.3,22 Segmental reflexes may be intact,
decreased, or absent, but pain sensation is preserved. Occa-
sionally tachycardia and tachypnea are present. Tachypnea
may result from diaphragmatic muscle weakness or second-
ary aspiration pneumonia. Increased lung sounds may also
be present on thoracic auscultation of dogs with secondary
aspiration pneumonia. Physical examination findings in one
affected cat consisted of hypothermia, lethargy, dehydration,
quadriplegia, and decreased segmental reflexes.5
Tetanus
In dogs with tetanus, involvement of the head and generalized
disease occurs more often than occurs in cats. A wound may or
522 SECTION 2  Bacterial Diseases

Skeletal Muscle

Normal Acetylcholine Exocytosis Disruption of Acetylcholine Exocytosis

at the Neuromuscular Junction at the Neuromuscular Junction

Ingestion
of toxin
Botulinum
toxin Light chain

s s

Heavy chain Absorption of

toxin from
intestine
Light chain of
botulinum toxin
cleaves syntaxin
and
interferes with release
of acetylcholine

Acetylcholine
Neurotransmitter Toxin into
vesicle Vesicle and Vesicle and blood vessel
SNARE
proteins terminal terminal
Synaptic fusion membranes membranes
VAMP/ complex forms fuse fail to fuse
Synaptobrevin
SNAP-25
Syntaxin

Synaptic Cleft

Heavy chain of
botulinum toxin
binds to receptor
on cell membrane

1A Acetylcholine Receptors Stimulated 1B Acetylcholine Receptors Not Stimulated

A (Normal Muscle Contraction) (Muscle Fails to Contract - Flaccid Paralysis)
FIGURE 54-2  Action of clostridial neurotoxins. A, Botulism Toxin.
Continued

may not be found. Dogs with localized forelimb tetanus have
caudal retraction of the limb with extension of both the elbow
and carpus.15 In dogs with generalized tetanus, a wrinkled fore-
head, erect ears, retracted lips (risus sardonicus), and prolapsed
third eyelids are almost always present, usually in association
with generalized muscle stiffness (see Figure 54-3, A). Miosis
may be present. It may be difficult or impossible to open the
mouth. Dogs that are ambulatory walk slowly with a stiff, stilted
gait and can have a wide-based or “sawhorse” stance; voluntary
movement of the head may occur slowly or appear difficult, and
the tail may be erect. Laryngeal spasm and hypersalivation occur
in approximately half of affected dogs.13,14 Reflexes are exag-
gerated or difficult to elicit. Severely affected dogs are anxious
and hypersensitive, develop opisthotonos, vocalize, tremble or
 CHAPTER 54  Tetanus and Botulism 523

Inhibitory
interneuron

Clostridium tetani

internalized
neurotoxin

Toxin into
blood vessel

TeNT

Synaptic Cleft

B
FIGURE 54-2, cont’d.  B, Tetanospasmin.

even have a seizure when stimulated, and may be hyperthermic
due to increased muscle activity (up to 111°F or 43.9°C).11 A
semicomatose state may even result. Abdominal palpation may
reveal an enlarged bladder that is difficult to express, or palpa-
tion may not be possible because of abdominal muscle spasm.
Hypoventilation may be present, or tetanic spasms may trigger
episodes of dyspnea. At least a third of dogs are bradycardic or
have bradyarrhythmias (which include atrioventricular block or
sinus arrest), with heart rates that range from 40 to 60 beats/
min; another third of dogs are tachycardic (>150 beats/min) or
have variable heart rates, which may reflect autonomic dysfunc-
tion or pain.11
524 SECTION 2  Bacterial Diseases

Cats with localized tetanus often have extensor rigidity of
a single limb, on which a wound may be identified. When the
forelimb is affected, spasm of the triceps muscle results in caudal
extension of the limb with carpal flexion (in contrast to dogs)
and elbow extension (Figure 54-3, B).20,23,24 The head and neck
may be deviated toward the affected limb.15 When disease gen-
eralizes, the contralateral limb and subsequently all four limbs
may be affected.20 Affected cats may be reluctant to move or
hyperesthetic. Lockjaw (trismus), protrusion of the third eyelid,
retracted lips (risus sardonicus), wrinkling of the forehead, or
opisthotonos can occur in cats with generalized tetanus. Seg-
mental reflexes may be exaggerated, or it may not be possible
to elicit reflexes because of severe muscle spasticity. Dyspnea,
tachypnea, and open-mouth breathing may occur.12 Cranial
nerve responses are usually normal. Miosis may be present.

Diagnosis
Diagnosis of tetanus and botulism in dogs and cats is most often
based on a consistent history and clinical abnormalities, exclusion
of the few other possible causes of disease (e.g., with assays for
myasthenia gravis in dogs with botulism), and sometimes electro-
diagnostic testing. The primary differential diagnoses for botulism
are acute polyradiculoneuritis and myasthenia gravis; paralytic
rabies should also be considered. There are few other disorders
other than strychnine poisoning that truly mimic tetanus. Serologic
assays have been used to detect an antibody response to botulinum
A toxins. Finally, specific assays that detect botulinum toxin in serum,
gastrointestinal contents, or food can be performed (Table 54-1).

B and/or increased liver enzyme activities and hyperbilirubinemia.
FIGURE 54-3  A, Image of a Weimaraner with generalized tetanus. The dog walked
with a stiff gait and was salivating profusely. Risus sardonicus is evident. B, Three-year-old
female domestic shorthair cat with localized tetanus that was seen for a 3-day history of
right thoracic limb lameness. Extension of the shoulder and elbow and partial flexion of
the carpal joint is present. (From Langner KFA, Schenk HC, Leithaeuser C, et al. Localised
tetanus in a cat. Vet Rec 2011;169:126.)
Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
Routine laboratory test results in animals with tetanus or botulism
are often normal, but stress hyperglycemia, mild to severe increases
in the activity of serum creatine kinase (usually <20,000 U/L but
sometimes >200,000 U/L, reference range 46-320 U/L), mildly
increased AST activity, and myoglobinuria may be present in animals
with tetanus.11,14 Dehydration may result in hemoconcentration or
prerenal azotemia. Animals with wounds or aspiration pneumonia
may have an inflammatory leukogram. Severe hyperthermia in dogs
with tetanus may lead to laboratory evidence of multiple organ dys-
function, with moderate to severe hypoalbuminemia, azotemia,
Thrombocytopenia and coagulation test abnormalities consistent
with DIC may also be present in these dogs.
CSF Analysis
CSF analysis in dogs and cats with botulism and tetanus
­typically shows no abnormalities.

TABLE 54-1
Laboratory Assays Available for Diagnosis of Botulism
Assay Specimen Type Target Performance
Mouse inoculation Feces, vomitus, intesti- Botulinum toxin Gold standard for diagnosis. False negatives can occur
­bioassay nal contents, serum, if toxin degradation occurs. Slow turnaround time
foodstuffs means diagnosis is generally retrospective. Laborious
and requires the use of laboratory animals.
ELISA toxin assays Feces, vomitus, intesti- Botulinum toxin Rapid and inexpensive. Standard ELISA toxin assays
nal contents, serum, have a lower sensitivity than the mouse inoculation
foodstuffs assay.
ELISA assays that detect Serum Anti–botulinum Negative antibody titers occur early in the course of
the antibody response toxin antibodies disease, so acute and convalescent titers must be
to botulinum toxin performed. Thus, diagnosis is retrospective.

A
B antibody response to tetanospasmin is not significant.
FIGURE 54-4  Radiographic findings in dogs with botulism (A) and tetanus (B). A, Lat-
eral thoracic radiograph from a 6-year-old female spayed Labrador retriever with botulism.
Megaesophagus is present. The white arrows delineate the dorsal and ventral esophageal
wall. B, Lateral thoracic radiograph from a 3-year-old female spayed English springer span-
iel with tetanus that was seen for vomiting, dysuria, respiratory distress, and a stiff gait.
Pneumothorax and a hiatal hernia (arrows) can be appreciated. Before the radiographs
were taken, an exploratory laparotomy had been performed because a foreign body was
suspected. The pneumothorax developed postoperatively for reasons that were unclear. A
foxtail wound was later found on the left distal pelvic limb between the digits. The dog was
hospitalized for 19 days, during which time all radiographic abnormalities resolved.
Diagnostic Imaging
Thoracic radiographs in dogs with botulism may be unremark-
able, or megaesophagus may be present (Figure 54-4, A). Evi-
dence of carrion bones are occasionally present in the stomach.
Changes consistent with aspiration pneumonia in dogs with
botulism or tetanus may also be apparent.11,22 Rarely, dogs with
tetanus have evidence of a hiatal hernia on thoracic radiogra-
phy, which appears as a soft tissue mass in the caudal thorax
(see Figure 54-4, B). Abdominal ultrasound examination may be
normal, or mild abdominal lymphadenopathy may be identified.
Electrodiagnostic Tests
Electromyography (EMG) and electroneurography can be useful
to support a diagnosis of botulism in some dogs (Table 54-2).
Sedation or anesthesia for electrophysiologic testing is gener-
ally not advisable in dogs with megaesophagus and aspiration
CHAPTER 54  Tetanus and Botulism 525
pneumonia, although EMG can be performed without anesthe-
sia.3 Electroneurography studies involve electrical stimulation of
one or more motor nerves and assessment of the resultant electri-
cal activity in the innervated muscle. Electroneurography findings
in dogs with botulism include normal F waves (late waves that
disappear in acute polyradiculoneuritis) and decreased magnitude
of the compound muscle action potential.3,22,25 Repetitive nerve
stimulation at low frequencies can result in decremental responses,
but repetitive nerve stimulation at high frequencies in dogs with
botulism has no effect on the muscle response.22 In humans with
botulism, an incremental response to repetitive nerve stimulation
occurs at high frequencies.26 Motor nerve conduction velocity
(MNCV) is normal or decreased. On EMG, the primary find-
ing is prolonged or increased insertional activity in botulism.3,22
Fibrillation potentials and positive sharp waves have also been
described.3,25 Animals with tetanus have had normal EMGs and
MNCVs as well as motor unit potentials and pathologic spon-
taneous muscle activity,14,20,21,24 but electrodiagnostic testing is
not specific for a diagnosis of tetanus and is usually not required.
Microbiologic Tests
Isolation and Identification
Attempts to culture C. tetani from wounds are not recommended
because the presence of C. tetani in a wound does not necessar-
ily imply toxin production. In addition, this is insensitive.11,13
Serologic Diagnosis
ELISA assays that detect the antibody response to botulinum neu-
rotoxin are available and were used to confirm a diagnosis of bot-
ulism in one dog.7 A convalescent specimen collected 3 weeks after
the onset of illness was positive, but specimens collected at the time
the dog was first evaluated and 10 days later were negative. Thus,
the diagnosis is generally retrospective with this method because
most dogs have recovered by the time seroconversion occurs. The
Toxin Detection
Botulinum toxin can be detected in feces, vomitus, gastrointesti-
nal contents collected at necropsy, carrion, or serum. Specimens
should be held at 4°C, marked with a warning label, and shipped
immediately to the laboratory. The most sensitive assay is a mouse
inoculation assay, in which untreated mice and mice treated with
serotype-specific antitoxin are inoculated with the specimen to
determine if the antitoxin is protective.27 However, several days
are required to run this assay. Toxin type–specific ELISA assays
are available for rapid detection of neurotoxin C, but standard
ELISA assays lack sensitivity when compared with mouse inocu-
lation. Research efforts have been focused on the development of
a variety of novel, rapid, highly sensitive, and inexpensive diag-
nostic assays that detect minute quantities of botulinum toxins.27
Pathologic Findings
There are no specific gross or histopathologic findings associ-
ated with botulism or tetanus. Evidence of complications such
as aspiration pneumonia, pulmonary thromboembolism, or
DIC may be present in dogs with tetanus.
Treatment and Prognosis
Specific Treatment
There is no readily available specific treatment for botulism
in dogs. In the United States, heptavalent equine antitoxin is
526 SECTION 2  Bacterial Diseases

TABLE 54-2
Pathophysiologic, Clinical, and Electrophysiologic Features of Botulism, Polyradiculoneuritis, and Myasthenia Gravis
Botulism Polyradiculoneuritis Acquired Myasthenia Gravis
Pathophysiology Inhibition of acetylcholine release by Inflammatory disorder of Antibody formation to acetylcho-
botulinum toxin nerve roots line receptors at the neuromus-
cular junction
Neurologic Decreased reflexes, progressive Decreased reflexes, progres- Normal reflexes, muscle weak-
­abnormalities ­tetraparesis, respiratory compro- sive tetraparesis, facial ness and rapid fatigue,
mise, autonomic signs, dysphonia, weakness, dysphonia, respi- ­megaesophagus
­megaesophagus ratory compromise, with or
without hyperesthesia
EMG Normal/subnormal Abnormal Normal
Electroneurography Decreased CMAP amplitudes, normal Decreased CMAP amplitudes, Usually normal CMAP ampli-
F-waves, decremental response to absent F-waves, usually tudes, decremental response to
repetitive nerve stimulation at low normal MNCV repetitive nerve stimulation at
frequency (and incremental response low frequency
at high frequency in humans)

Modified from Uriarte A, Thibaud JL, Blot S. Botulism in 2 urban dogs. Can Vet J 2010;51:1139-1142.
CMAP, Compound muscle action potential; MNCV, Motor nerve conduction velocity.

TABLE 54-3 The efficacy of tetanus antitoxin is controversial, and the opti-
mal dose is not known. In one study, early IV administration of
Suggested Dosages of Medication for Specific Treatment equine tetanus antitoxin did not influence disease progression
of Tetanus or mortality as opposed to administration later in the course of
disease.11
Interval Duration For animals with tetanus, treatment with antimicrobials that
Drug Dose Route (hours) (days) have activity against C. tetani is also recommended. This is not
Metronidazole 15 mg/kg IV, PO 12 10 indicated for animals with botulism, because ingested toxin is
preformed. Appropriate antimicrobials for treatment of tetanus
Tetanus equine 100-1000 IV, IM* NA Once
include intravenous metronidazole or penicillin G. Metronida-
antitoxin units/kg NA Once
zole is preferred because of its improved penetration into puru-
500 units SC near
lent material and the fact that penicillin can antagonize GABA.
wound site
Studies in human patients have also shown improved outcome
Human tetanus 500 units IM near IM Once with metronidazole as opposed to procaine penicillin.29 Once
immune wound if antitoxin has been administered and spasms are controlled (see
globulin found Supportive Care), wounds should also be debrided or flushed
extensively with hydrogen peroxide solution. Earlier appropri-
NA, Not applicable.
ate antimicrobial treatment and wound debridement may not
*IV use is preferred. Administer 0.1 mL intradermally as a test dose be-
fore infusion. The full dose should then be given slowly over 10 minutes. influence progression or mortality.11

available from the Centers for Disease Control and Prevention
for treatment of human botulism. However, its availability is
tightly controlled, so it may be impossible to obtain for treat-
ment of dogs or cats.28 Tetanus antitoxin is more widely avail-
able and should be administered as a single dose IV (equine
antitoxin) or IM (human antitoxin) after an initial intradermal
test dose to evaluate for allergic reactions (Table 54-3). Dogs
that react to the test dose should be premedicated with diphen-
hydramine and a low dose of dexamethasone. Adverse reactions
to antitoxin include vomiting, retching, or tachypnea and may
occur even in premedicated dogs.11 Reduction in the rate of
antitoxin administration or discontinuation of treatment leads
to resolution of clinical signs. Administration of antitoxin does
not reverse existing paralysis because it is unable to enter nerve
terminals, but it may neutralize free toxin. A small amount of
antitoxin can be administered locally if a wound is identified.
Supportive Care
For both tetanus and botulism, supportive care is an essential
part of management. The extent of care required depends on
the severity of illness. Intravenous fluids, soft bedding, frequent
turning to prevent decubital ulcers, application of ocular lubri-
cant to prevent corneal ulceration, bladder expression or uri-
nary catheterization (ideally intermittent), enemas to relieve
constipation, and nutritional support in the form of hand feed-
ing, feeding tubes, or total parenteral nutrition may be required.
Force feeding is not recommended because it is unlikely to
satisfy caloric needs and can contribute to the development of
aspiration pneumonia. Occasionally (<10% of affected dogs
with tetanus), mechanical ventilation is required to support res-
piration.11 For dogs with botulism, antimicrobial drugs should
only be used if infection develops (e.g., bacterial pneumonia or
urinary tract infection). Aminoglycosides should be avoided
because they may contribute to neuromuscular blockade.

Dogs or cats with generalized tetanus should be placed in
a quiet, dimly lit environment away from excessive visual or
auditory stimuli. Cotton balls can be used as earplugs. Seda-
tives and/or muscle relaxants such as acepromazine, diazepam,
midazolam, or methocarbamol may be useful. Severely affected
animals may require a constant-rate infusion of drugs such as
pentobarbital, phenobarbital, or propofol. Barbiturates are no
longer recommended for treatment of tetanus in humans; ben-
zodiazepines are preferred. If human disease is severe, drugs that
cause neuromuscular blockade such as vecuronium are used, but
this necessitates mechanical ventilation.30 Methocarbamol often
appears ineffective and requires frequent administration.11,13
Benzodiazepines are preferred to methocarbamol because they
are GABA agonists and also provide some sedation. Success-
ful control of muscle spasms relieves pain and hyperthermia.
If used, opioids should be administered with caution because
they may contribute to constipation or gastrointestinal adverse
effects. The use of fans and cool fluids may also be beneficial
for dogs with tetanus-induced heat stroke. Administration of
atropine may be required for animals with bradyarrhythmias
(HR <60 beats/min). Rarely, upper respiratory obstruction has
necessitated tracheostomy,14 or bradyarrhythmias have required
temporary pacemaker placement.11
Prognosis
Provided complications do not occur, prognosis is generally
good for animals with tetanus or botulism. The overall 28-day
survival rate for dogs with tetanus in one study was around
80%,11 whereas in another study of 20 dogs it was only 50%.13
A classification system has been developed to assess the sever-
ity of tetanus in dogs for the purpose of prognostication (Table
54-4).11 The survival rate for dogs that progressed to tetanus
severity classes I or II was 100%, but it dropped to 60% for
dogs that progressed to tetanus severity class III or IV.11 How-
ever, at the time of initial evaluation, only 11% of 37 dogs were
in classes III or IV, and dogs progressed to higher classes unpre-
dictably, with 47% of the dogs categorized in class III or IV
when the highest recorded class was assigned. Dogs with severe
tetanus are more likely to develop complications such as aspira-
tion pneumonia or multiple organ dysfunction than dogs with
mild tetanus. In a few dogs that recover (<10% of dogs that
develop tetanus), intermittent localized muscle spasm continues
to occur for many months, which may be noticed by the owner
during activities such as sleeping or eating.11
Immunity and Vaccination
Given the relative rarity of tetanus and botulism in dogs and
cats, vaccination is not recommended for these species. Recur-
rent tetanus or botulism has been documented in human
patients, so recovery from natural disease does not necessarily
result in protection from future episodes of disease. Thus, pre-
ventative measures should be discussed with owners of pets that
recover from tetanus or botulism.
Prevention
Prevention of botulism involves limiting access of cats and dogs
to carrion and feeding heat-processed commercial diets that are
unspoiled. Although botulinum toxin can be denatured through
boiling, spores can survive this treatment. Thus, cooked food
should not be allowed to stand for long periods before it is fed
CHAPTER 54  Tetanus and Botulism 527
TABLE 54-4
Tetanus Severity Classification System for Dogs
Class Clinical Signs
I Absence of class II, III or IV signs and any or all of:
• Miosis, enophthalmos, risus sardonicus, erect
ears, or trismus
• Hypersensitivity to noise, light or touch
• Ambulatory
II Absence of class III or IV signs and any or all of:
• Dysphagia
• Stiff gait, sawhorse stance, erect tail
• Ambulatory
III Class I or class II signs, absence of class IV signs, and
any or all of:
• Recumbency
• Muscle fasciculations or spasms
• Seizures
IV Class I, II, or III signs and any or all of:
• Bradycardia (HR ≤60 beats/min) or bradyar-
rhythmia
• Sinus tachycardia (HR ≥140 beats/min) or
tachyarrhythmia
• Labile hypertension or hypotension (mean
arterial blood pressure ≤60 mm Hg or ≥ 130
mmHg, or systolic arterial blood pressure ≤80
mm Hg or ≥150 mm Hg)
• Periods of apnea or respiratory arrest
Modified from Burkitt JM, Sturges BK, Jandrey KE, et al. Risk factors
associated with outcome in dogs with tetanus: 38 cases (1987-2005).
J Am Vet Med Assoc 2007;230:76-83.
to pets. The spores of C. botulinum are inactivated by pressure-
cooking foods.
For tetanus, control of (1) roaming, (2) exposure to plant
awns, and (3) aggressive interactions with other animals may
reduce the chance that wounds develop. If wounds are noticed,
they should be cleaned promptly with soap and water. Surgical
instruments should always be sterilized properly before use so
that tetanus spores are inactivated, and needles should never be
reused. Tetanus spores can be inactivated with iodine, glutaral-
dehyde, hydrogen peroxide, or autoclaving at 121°C and 15 psi
for 15 minutes.
Public Health Aspects
Animals with botulism or tetanus cannot transmit these condi-
tions directly to humans who handle them. Nevertheless, feces,
tissues, or body fluids from animals with botulism should be
handled with care and marked with warning labels before they
are submitted to the laboratory because the toxin is so potent.
The main risk that dogs with tetanus pose to humans is through
bite wounds, which can occur when owners attempt to admin-
ister medications.
Three forms of botulism occur in people: infant botulism,
wound botulism, and food botulism.26 Infant botulism occurs as a
result of local production of botulinum toxin within the intestinal
tract by C. botulinum in the absence of the competing intestinal
528 SECTION 2  Bacterial Diseases
microflora that are normally found in adults. Wound botulism
occurs almost exclusively as a result of injection of contaminated
heroin into the skin by intravenous drug users. Botulinum toxin
preparations are used to treat conditions such as strabismus,
esophageal achalasia, cervical dystonia, axillary hyperhydrosis
(excessive sweating), or blepharospasm. There are rare reports
of iatrogenic botulism after such use.31 Life-threatening iatro-
genic botulism has also occurred after use of botulinum prepara-
tions that were unlicensed for cosmetic treatment of wrinkles.32
CASE EXAMPLE
Signalment: “Jenny,” a 6-year-old female spayed Labrador
retriever from Winters in northern California
History: Jenny was brought to the University of California,
Davis, veterinary emergency service early in the morning for a
2-hour history of inability to stand. Her owners awoke to find
her in another room, unable to get up for her morning walk.
She appeared anxious and was breathing with increased
effort, but was still wagging her tail. She also did not want
to eat breakfast. There had been no known history of travel,
toxins, trauma, or tick exposure. Over the past 2 months,
she had been treated with prednisone (1 mg/kg PO q24h
for 5 days, followed by 0.74 mg/kg PO q24h thereafter) for
allergic dermatitis. Three weeks before the onset of illness she
was treated with a short course of cephalexin after an aural
hematoma was repaired. Jenny had been regularly vaccinated
with rabies, distemper, hepatitis, and parvovirus vaccines.
Physical Examination:
Body Weight: 27.2 kg
General: Bright, alert, responsive, and hydrated. Laterally
recumbent and anxious. HR = 140 beats/min, T = 101°F,
panting.
Integument, Eyes, Ears, Nose, and Throat: Sutures were
present on the pinna of the right ear from the recent aural
hematoma repair. Moderate dental calculus and gingivitis
was present. There were no other abnormalities noted.
Musculoskeletal: BCS 5/9. The dog was unable to use the
pelvic limbs to bear weight.
Cardiovascular, Respiratory, Gastrointestinal, Genitouri-
nary, and Lymph Nodes: No clinically significant abnor-
malities were present.
Neurologic Examination:
Mentation: Appropriate, anxious.
Gait/Posture: The dog was nonambulatory, but voluntary
movement was present in all four limbs. She was unable to
stand and support weight, but could crawl and shuffle. She
attempted to bear weight on her thoracic limbs and could
maintain sternal recumbency. When supported to stand, the
pelvic limbs collapsed and the thoracic limbs were minimally
able to bear weight.
Cranial Nerves: Slightly reduced palpebral reflexes and gag
reflex. Absent corneal reflexes. No other deficits were noted.
Postural Reactions: These were difficult to assess because of
the dog’s inability to stand, but appeared decreased.
Significant concerns have been raised about the possible use of
botulinum toxin as an agent of bioterrorism. This has stimulated
considerable research on the disease, which is otherwise very rare.
Tetanus in humans is rare in developed countries, because
most children are immunized against the disease. When it
occurs, it is most often in people older than 60 years of age
because of waning immunity.30 Worldwide, the vast majority
of cases occur in neonates from sub-Saharan Africa because of
lack of immunization.
Segmental Reflexes: Reduced muscle tone was present in the
thoracic limbs. Triceps, patella, and gastrocnemius reflexes
were decreased. Reduced thoracic limb withdrawal reflexes
were noted, but pelvic limb withdrawal reflexes were intact.
Panniculus and perineal reflexes were also intact. Pain
perception remained intact.
Spinal Palpation: No apparent pain was present on palpation
or cervical manipulation.
Neuroanatomic Location: Neuromuscular disease.
Laboratory Findings:
CBC:
HCT 41.0% (40%-55%)
MCV 69 fL (65-75 fL)
MCHC 36.6 g/dL (33-36 g/dL)
WBC 5800 cells/µL (6000-13,000 cells/µL)
Neutrophils 3671 cells/µL (3000-10,500 cells/µL)
Lymphocytes 1769 cells/µL (1000-4000 cells/µL)
Monocytes 197 cells/µL (150-1200 cells/µL)
Eosinophils 145 cells/µL (0-1500 cells/µL)
Platelets clumped but adequate (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 146 mmol/L (145-154 mmol/L)
Potassium 3.9 mmol/L (3.6-5.3 mmol/L)
Chloride 107 mmol/L (108-118 mmol/L)
Bicarbonate 21 mmol/L (16-26 mmol/L)
Phosphorus 2.3 mg/dL (3.0-6.2 mg/dL)
Calcium 11.4 mg/dL (9.7-11.5 mg/dl)
BUN 15 mg/dL (5-21 mg/dL)
Creatinine 0.9 mg/dL (0.3-1.2 mg/dL)
Glucose 105 mg/dL (64-123 mg/dL)
Total protein 7.1 g/dL (5.4-7.6 g/dL)
Albumin 4.2 g/dL (3.0-4.4 g/dL)
Globulin 2.9 g/dL (1.8-3.9 g/dL)
ALT 64 U/l (19-67 U/L)
AST 25 U/L (19-42 U/L)
ALP 129 U/L (21-170 U/L)
Creatine kinase 57 U/L (51-399 U/L)
GGT 4 U/L (0-6 U/L)
Cholesterol 204 mg/dL (135-361 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.025; pH 7.0, trace protein (SSA), 1+ bilirubin,
no hemoprotein, glucose, ketones, WBC/HPF, RBC/HPF,
bacteria or crystals in the sediment. Many lipid droplets
were present.

Imaging Findings:
Thoracic Radiographs: The cardiopulmonary structures were
within normal limits. There were mineral opacities within the
stomach. There was a small amount of gas in the midthoracic
esophagus.
Abdominal Ultrasound Examination: Mild mesenteric
lymphadenopathy was identified. No other abnormalities
were present.
Neuromuscular Testing: Edrophonium response test for
myasthenia gravis: negative
Treatment: Jenny was admitted to the intensive care
unit and treated with supportive care that included IV
fluids (lactated Ringer’s solution with 20 mEq/L KCl at
75 mL/hr), soft bedding, intermittent manual bladder
expression, physical therapy (massage and passive range-
of-motion exercises), and frequent turning. Treatment
with prednisone was discontinued. Respiratory function
was monitored continuously by observation, serial
examination, and serial venous pCO2 measurements.
After repeated inspections for ticks, fipronil was applied
in case the signs resulted from tick paralysis. Serum was
submitted for an acetylcholine receptor antibody titer,
and feces were submitted for a botulinum toxin assay.
Over the subsequent 12 to 18 hours, Jenny became
anxious after she was fed canned food and began to
retch and hypersalivate. Thoracic radiographs were
repeated and showed megaesophagus (see Figure 54-4,
A). Severe tachycardia also developed (HR = 210 beats/
min). An ECG showed ventricular tachycardia followed
by supraventricular tachycardia. Systolic blood pressure
was 145 mm Hg. Acepromazine was administered (0.01
mg/kg IV once), after which her heart rate decreased
to 90 beats/min, with a normal sinus rhythm. A second
neurologic examination showed progression of disease
with reduced cervical tone and an inability to hold up the
head, tetraplegia with absence of all segmental reflexes,
and a reduced tail wag. The owners were warned that
mechanical ventilation may be needed should the disease
progress. Over the course of the next day, dysphonia and
a mild cough developed, and Jenny regurgitated several
times. Gastrostomy or jejunostomy tube feeding or
SUGGESTED READINGS
Burkitt JM, Sturges BK, Jandrey KE, et al. Risk factors associated with
outcome in dogs with tetanus: 38 cases (1987-2005). J Am Vet Med
Assoc. 2007;230:76-83.
Cai S, Singh BR, Sharma S. Botulism diagnostics: from clinical symp-
toms to in vitro assays. Crit Rev Microbiol. 2007;33:109-125.
Chertow DS, Tan ET, Maslanka SE, et al. Botulism in 4 adults following
cosmetic injections with an unlicensed, highly concentrated botuli-
num preparation. JAMA. 2006;296:2476-2479.
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1884;10:842-844.
2. Van Ermengem EP. Über einen neuen anaeroben Bacillus und
seine Beziehung zum Botulismus. Z Hyg Infektionskrankh.
1897;26:1-56.
CHAPTER 54  Tetanus and Botulism 529
parenteral nutrition were offered to the owner, but were
declined. Four days after admission, improvement in the
triceps reflex was noted, and on day 5, some voluntary
movement was noted in the pelvic and thoracic limbs. At
that time Jenny was successfully fed meatballs of canned
food by hand. She was kept in sternal recumbency for 30
minutes after eating to minimize further aspiration. On
day 7, the acetylcholine receptor antibody and botulinum
toxin assay results became available.
Serum acetylcholine receptor antibody titer: 0.05 nmol/L (refer-
ence range, <0.6 nmol/L)
Botulinum toxin C mouse bioassay (fecal specimen): Positive
for type C botulinum toxin. An extract from the fecal speci-
men was lethal in the bioassay. The effects were eliminated
by heating the extract at 80°C for 10 minutes, which indi-
cated that the toxic effects were due to a heat-labile toxin.
Mice were also protected from the effects by anti–type
C botulinum antisera but not by anti–type A botulinum
antisera.
Diagnosis: Botulism.
Outcome: On day 8 after hospitalization, thoracic radiographs
showed resolution of megaesophagus and no evidence of
pneumonia. By day 10, Jenny began to shuffle along the floor.
All segmental reflexes had returned but remained reduced.
The dog was discharged from the hospital with instructions
for continuation of care in the home environment. By day 15,
Jenny’s owners reported that her normal bark had returned,
and she could walk 20 steps and defecate and urinate
unassisted. A month after discharge she was reexamined
and no abnormalities were detected.
Comments: The main differential diagnoses considered for
this dog were myasthenia gravis and botulism. Initially,
polyradiculoneuritis was also considered, but the subsequent
development of megaesophagus was not consistent
with this disorder. Ultimately, a diagnosis of botulism was
confirmed through assay of the stool for botulinum toxin. A
history of carrion ingestion was not known, but the dog lived
in a semirural area, and the presence of mineralized stomach
contents suggested that it had occurred. Prednisone
administration may have contributed to polyphagia and
ingestion of carrion by this dog.
3. Barsanti JA, Walser M, Hatheway CL, et al. Type C botulism in
American Foxhounds. J Am Vet Med Assoc. 1978;172:809-813.
4. Darke PG, Roberts TA, Smart JL, et al. Suspected botulism in fox-
hounds. Vet Rec. 1976;99:98-99.
5. Elad D, Yas-Natan E, Aroch I, et  al. Natural Clostridium botu-
linum type C toxicosis in a group of cats. J Clin Microbiol.
2004;42:5406-5408.
6. Sobel J, Tucker N, Sulka A, et al. Foodborne botulism in the United
States, 1990-2000. Emerg Infect Dis. 2004;10:1606-1611.
7. Bruchim Y, Steinman A, Markovitz M, et al. Toxicological, bacte-
riological and serological diagnosis of botulism in a dog. Vet Rec.
2006;158:768-769.
8. Wallace V, McDowell DM. Botulism in a dog—first confirmed case
in New Zealand. N Z Vet J. 1986;34:149-150.
9. Farrow BR, Murrell WG, Revington ML, et al. Type C botulism in
young dogs. Aust Vet J. 1983;60:374-377.
530 SECTION 2  Bacterial Diseases
10. Doutre MP. 2nd case of botulism type D in the dog in Senegal. Rev
Elev Med Vet Pays Trop. 1983;36:131-132.
11. Burkitt JM, Sturges BK, Jandrey KE, et al. Risk factors associated
with outcome in dogs with tetanus: 38 cases (1987-2005). J Am Vet
Med Assoc. 2007;230:76-83.
12. Sykes JE. Unpublished observations, 2012.
13. Bandt C, Rozanski EA, Steinberg T, et  al. Retrospective study
of tetanus in 20 dogs: 1988-2004. J Am Anim Hosp Assoc.
2007;43:143-148.
14. Adamantos S, Boag A. Thirteen cases of tetanus in dogs. Vet Rec.
2007;161:298-302.
15. Malik R, Church DB, Maddison JE, et al. Three cases of local teta-
nus. J Small Anim Pract. 1989;30:469-473.
16. Meunier FA, Lisk G, Sesardic D, et al. Dynamics of motor nerve
terminal remodeling unveiled using SNARE-cleaving botulinum
toxins: the extent and duration are dictated by the sites of SNAP-
25 truncation. Mol Cell Neurosci. 2003;22:454-466.
17. Goldhammer MA, Chapman PS, Grierson JM. Coxofemoral luxa-
tion in a Border collie as a complication of a Clostridium tetani
infection. J Small Anim Pract. 2008;49:159-162.
18. Langner KF, Schenk HC, Leithaeuser C, et al. Localised tetanus in
a cat. Vet Rec. 2011;169:126.
19. Lee EA, Jones BR. Localised tetanus in two cats after ovariohyster-
ectomy. N Z Vet J. 1996;44:105-108.
20. Polizopoulou ZS, Kazakos G, Georgiadis G, et al. Presumed local-
ized tetanus in two cats. J Feline Med Surg. 2002;4:209-212.
21. Tomek A, Kathmann I, Faissler D, et al. [Tetanus in cats: 3 case
descriptions]. Schweiz Arch Tierheilkd. 2004;146:295-302.
22. Uriarte A, Thibaud JL, Blot S. Botulism in 2 urban dogs. Can Vet J.
2010;51:1139-1142.
23. Malik R, Simpson DJ, Church DB. What is your diagnosis? Teta-
nus. J Small Anim Pract. 1998;39(217):252.
24. De Risio L, Gelati A. Tetanus in the cat—an unusual presentation.
J Feline Med Surg. 2003;5:237-240.
25. van Nes JJ, van der Most van Spijk D. Electrophysiological evi-
dence of peripheral nerve dysfunction in six dogs with botulism
type C. Res Vet Sci. 1986;40:372-376.
26. Reddy P, Bleck TP. Clostridium botulinum (botulism). In:
Mandell GL, Bennett JE, Dolin R, eds. Principles and Prac-
tice of Infectious Diseases. 7th ed. Philadelphia, PA: Elsevier;
2010:3097-3102.
27. Cai S, Singh BR, Sharma S. Botulism diagnostics: from clinical
symptoms to in vitro assays. Crit Rev Microbiol. 2007;33:109-125.
28. Investigational heptavalent botulinum antitoxin (HBAT) to
replace licensed botulinum antitoxin AB and investigational
botulinum antitoxin E. MMWR Morb Mortal Wkly Rep.
2010;59:299.
29. Ahmadsyah I, Salim A. Treatment of tetanus: an open study to
compare the efficacy of procaine penicillin and metronidazole. Br
Med J (Clin Res Ed). 1985;291:648-650.
30. Reddy P, Bleck TP. Clostridium tetani (tetanus). In: Mandell GL,
Bennett JE, Dolin R, eds. Principles and Practice of Infectious Diseases.
7th ed. Philadelphia, PA: Elsevier; 2010:3091-3096.
31. Coban A, Matur Z, Hanagasi HA, et  al. Iatrogenic botulism
after botulinum toxin type A injections. Clin Neuropharmacol.
2010;33:158-160.
32. Chertow DS, Tan ET, Maslanka SE, et al. Botulism in 4 adults fol-
lowing cosmetic injections with an unlicensed, highly concentrated
botulinum preparation. JAMA. 2006;296:2476-2479.
CHAPTER 55
Yersinia pestis (Plague) and Other Yersinioses
Jane E. Sykes and Bruno B. Chomel
Overview of Plague
First Described: Descriptions of plague date back to around
1300 bc. The causative agent was isolated in 1894, in Hong
Kong (Alexandre Yersin).1
Cause: Yersinia pestis, a gram-negative coccobacillus (family
Enterobacteriaceae)
Affected Hosts: Y. pestis causes disease in rodents, rabbits,
wild carnivores, domestic dogs and cats, and humans.
Goats, deer, antelope, camelids, and nonhuman primates
can also develop illness.
Geographic Distribution: Southwestern United States; foci
also exist in Asia, Africa, and South America
Major Mode of Transmission: Flea-borne, via an enormous
variety of rodent flea species; ingestion of infected
rodents or rabbits by cats and dogs
Major Clinical Signs: Fever, lethargy, inappetence, lymphade-
nopathy, subcutaneous abscesses
Differential Diagnoses: Tularemia, cat bite abscesses, Strepto-
coccus spp. infections, feline infectious peritonitis
Human Health Significance: A small percentage (10%) of
human plague cases result from exposure to infected cats,
through bites, scratches, or respiratory droplet inhalation
from cats with pneumonic plague. Dogs and cats can also
expose humans through the carriage of infected fleas into
households (especially sleeping areas).
Etiology and Epidemiology
The genus Yersinia includes 11 different species, three of which
are pathogenic in humans, cats, and dogs: Yersinia pestis and
the enteropathogenic species Yersinia pseudotuberculosis and
Yersinia enterocolitica. Yersinia spp. are nonmotile, gram-neg-
ative coccobacilli that belong to the family Enterobacteriaceae.
Only Y. pestis is transmitted by arthropod vectors.
Yersinia pestis causes plague, which is maintained in wild
burrowing rodents in rural regions of Asia, Africa, and the
Americas (Figure 55-1). There is no plague in Australia, and the
last case of plague in Europe was reported after World War II.
The organism is transmitted between rodents by rodent fleas;
humans, dogs, and cats are usually accidental and “dead-end”
hosts (although direct transmission can occur through aerosols
in humans and cats with pneumonic disease). Fleas become
infected when they feed on heavily bacteremic rodents. The
organism replicates in the flea intestine and creates a blockage
of the intestinal tract, which causes the “starved” flea to repeat-
edly and aggressively feed and regurgitate bacteria into the bite
site of a new host.2 A large number of flea species can transmit
Y. pestis, but some flea species, such as the Oriental rat flea
(Xenopsylla cheopis) worldwide and the ground squirrel flea
(Oropsylla montana) in the western United States are partic-
ularly efficient vectors. Although nowhere near as efficient as
these two fleas as a vector, the cat flea (Ctenocephalides felis)
can infest rodent reservoirs and also has the potential to trans-
mit Y. pestis.3 Y. pestis attains very high concentrations in
rodent blood (108 organisms/mL) before death of the rodent
occurs, which allows fleas to acquire sufficient numbers of
bacteria for efficient transmission to other rodents.4 Fleas can
remain infected for more than a year, which permits transmis-
sion long after the death of an infected rodent.
Plague is transmitted to cats or dogs when they ingest infected
mammals (especially rodents or rabbits) or less commonly, after
the bite of a rodent flea. Humans are infected by (1) the bite of an
infected flea; (2) direct contact with contaminated tissues (such
as handling dead rodents, rabbits, or other dead wild mamma-
lian carnivores); or rarely (3) inhalation of respiratory secretions
by infected animals, especially cats (Figure 55-2). Death of a
rodent from plague can cause the rodent’s fleas to bite humans
and other animals in a search for an alternative blood meal.
In the United States, plague in humans, dogs, and cats is
now uncommon, although it is likely underrecognized in dogs
and cats. Cats are considerably more susceptible to disease than
dogs. Affected cats can be of any age, breed, and sex, with a
mean age of 3 years in one case series.5 Most plague cases in
humans and cats occur in the southwestern United States, par-
ticularly northern New Mexico, but also southern Colorado,
northern Arizona, and to a lesser extent California, southern
Oregon, and western Nevada (especially in the Sierra Mountain
ranges) (Figure 55-3). The probability of an area in the United
States being a high-risk plague habitat increases with elevation
up to 2300 meters and declines as elevation increases there-
after.6 Rodent or rabbit species associated with human cases
include white-tailed antelope squirrels, ground squirrels, rock
squirrels, cottontail rabbits, jack rabbits, prairie dogs, deer mice,
Colorado chipmunks, and wood rats.2 The prevalence of anti-
bodies to Y. pestis in a survey of 4115 dogs and 466 cats from
California in the mid-1990s was less than 5% in both species.7
In general, plague has a seasonal distribution that correlates
with the timing of epizootics that lead to a die-off of susceptible
rodents. In the United States, most plague cases occur from Feb-
ruary through August, when rodents and their fleas are most
active and humans and their companion animals are more likely
to be outdoors. In general, the organism does not survive well in
the environment outside infected hosts, with survival less than
531
532 SECTION 2  Bacterial Diseases
FIGURE 55-1  Worldwide distribution of plague endemic areas. (Data compiled from the World Health Organization, Centers for Disease Control and Prevention, and other sources.
From Centers of Disease Control and Prevention. Prevention of plague: recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 1996;45:1-15.
In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases, 7th ed. Philadelphia, PA, Churchill Livingstone, Elsevier, 2011.)
Flea Ingestion
bite
Bites, Scratches
Aerosols
(pneumonic plague)
Rare
Aerosol
(pneumonic
plague)
FIGURE 55-2  The role of the cat in transmission of Yersinia pestis to humans. Most
humans are infected by direct contact with wildlife reservoir hosts or a bite from an
infected flea, but infections can also occur as a result of cat bites or direct exposure to cats
that have pneumonic plague. Dogs can also be infected but are less susceptible than cats to
disease and shedding. Direct aerosol transmission from one human to another is rare and
only occurs in humans that have terminal pneumonic plague.
72 hours on hard surfaces.8 However, Y. pestis survived at least
24 days in blood-contaminated soil that was protected from UV
light.9 The extent to which environmental survival of Y. pestis
contributes to the geographic distribution and epidemiology of
the disease requires further clarification.
Clinical Features
Signs and Their Pathogenesis
After inoculation into the mammalian host by fleas, Y. pestis
is taken up by mononuclear cells, in which it survives, repli-
cates, and produces F1 capsular envelope antigen. The envelope
allows it to further resist phagocytosis. Cats and dogs usually
become infected after they ingest bacteremic rodents or other
wildlife species that may have died of plague. In this situation,
the organism has already produced a capsule and disseminates
more rapidly than occurs after flea-borne transmission. Organ-
isms are carried by lymphatics to regional lymph nodes. In more
than half of affected cats, this results in an intense neutrophilic
inflammatory response, variable fever, inappetence, and forma-
tion of the bubo, which is an enlarged and tender lymph node
that occasionally forms an abscess and drains purulent material.
This form of disease is known as bubonic plague and is the least
fatal form of the disease. The location of the bubo(es) depends
on the initial site of infection. In cats that ingest infected rodents,
the mandibular or retropharyngeal lymph nodes are usually
involved, and affected cats shed bacteria from the oropharynx.
Less often (around 20% of cats with bubonic plague), other
peripheral lymph nodes are involved; this may follow flea-borne
transmission that occurs at sites distant to the head.5
In some cats, bubo formation is accompanied by bacteremia,
with associated endotoxemia and sepsis, and formation of hem-
orrhagic, neutrophilic, and necrotizing lesions in many organs.
Disseminated intravascular coagulation and multiple organ
failure can then ensue. Signs of severe sepsis or septic shock in
affected cats include tachypnea, vomiting, tachycardia or brady-
cardia, and weak pulse quality. In some situations, bacteremia
and multiple organ involvement occurs in the absence of identifi-
able buboes (septicemic form), which has a high mortality rate.
Pneumonic plague occurs in about 10% of affected cats as a result
of hematogenous spread of bacteria to the lungs or, less often, by
inhalation of organisms and can result in dyspnea and cough.
The incubation period for plague is estimated to be 1 to 6 days,
with shorter incubation periods after ingestion or inhalation of
the organism, and death can occur in as few as 2 to 3 days in the
absence of appropriate treatment; occasionally the clinical course
extends for 2 to 3 weeks. Some (<20%) of cats show no signs of
illness, or there may be transient fever, decreased appetite, and
recovery. Transient febrile illness is also common in dogs.
Physical Examination Findings
Physical examination in cats with plague reveals lethargy (82%
of cats), fever (71% of cats; mean rectal temperature of 105.1°F
[40.5°C]), dehydration, a thin body condition, and lymphade-
nopathy. Lymphadenopathy most often involves the mandib-
ular nodes. In one case series, 33% of 67 cats with bubonic
 CHAPTER 55  Yersinia pestis (Plague) and Other Yersinioses
plague had bilateral lymphadenopathy, 32% had left-sided
lymphadenopathy, and 13% had right-sided lymphadenopathy;
in the remainder of cats, the location was not known.5 Bilat-
eral disease occurred only when the mandibular and cervical
nodes were involved. Lymph nodes may be markedly enlarged
(up to 8 cm, with a mean of around 4 cm); may be edematous,
abscessated, and painful on palpation; and may drain purulent
material. Drainage was reported in 6 (15%) of 40 cats with
bubonic plague. Dyspnea or tachypnea can be present in cats
with pulmonary involvement or possibly as a result of painful
lymphadenopathy. Other reported physical examination abnor-
malities in cats with plague include ocular or nasal discharge
(14% of cats) and abscesses that involve sites other than the
lymph nodes, such as the sublingual region or the tongue, oral
cavity, face, limbs, or trunk. Physical examination findings in
dogs suspected to have plague have included fever, lethargy,
mandibular or cervical bubo formation, oral ulceration and
ptyalism, and cough.10
FIGURE 55-3  Approximate distribution of human plague cases in western United
States, 1970 to 2012. Each dot represents a case, with the dot placed randomly in the
county of exposure. (Modified from Dennis DT, Mead PS. Yersinia species, including plague.
In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas and Bennett’s Principles and
Practice of Infectious Diseases, 7th ed. Philadelphia, PA, Churchill Livingstone, Elsevier,
2011. Data from 2007-2012 collated from ProMed-Mail reports, http://www.promedmail.
org/. Last accessed February 5, 2013).
TABLE 55-1
Diagnostic Assays Currently Available for Plague in Dogs and Cats
Assay Specimen Type Target
Culture Lymph node ­aspirates, Yersinia pestis
blood, tissues
­collected at necropsy
Cytology Lymph node aspirates Y. pestis organisms Low sensitivity. Recent antibiotic treatment may cause false-
(bipolar staining bacilli) negative results.
Direct IFA Lymph node aspirates, Y. pestis F1 antigen
tissues collected at
necropsy
Serology Serum Antibodies to Yersinia
pestis
533
Diagnosis
Diagnosis of plague is based on a history of residence in an
endemic area as well as contact with, or predation of, wild
mammals (especially rodents or lagomorphs). Duration of ill-
ness, physical examination findings, and lymph node aspirate
cytology also support the diagnosis. There is a relative lack of
information available on laboratory abnormalities in dogs and
cats with plague because the disease is rare. Ultimately, a diag-
nosis of plague can only be confirmed by culture, direct fluo-
rescent antibody testing, and/or serologic testing. Confirmation
of the diagnosis may ultimately take weeks, so a high level of
suspicion for the disease is needed in endemic areas.
Laboratory Abnormalities
Hematologic abnormalities in naturally infected cats most often
consist of mild to moderate neutrophilia, usually with a mild to
moderate left shift, and occasionally lymphopenia and mono-
cytopenia.5 Mild leukopenia can occur in cats with pneumonic
plague. Platelet counts have not been widely reported in natu-
rally infected cats but may be low in cats with severe sepsis. The
serum biochemistry panel may show hypoalbuminemia, hyper-
glycemia, azotemia, electrolyte abnormalities, and increased
liver enzyme activities.11
Diagnostic Imaging
Plain Radiography
Findings on thoracic radiography in cats with pneumonic plague
have not been described in detail, but include diffuse interstitial,
alveolar, and often nodular lung opacities.
Sonographic Findings
Abdominal ultrasound findings in cats with plague have
not been reported. Based on pathologic findings, they might
include abdominal lymphadenomegaly, splenomegaly, and
hepatomegaly.
Microbiologic Tests
Diagnostic assays currently available for plague in dogs and cats
are described in Table 55-1.
Performance
Recent antibiotic treatment may cause false-negative results.
May take 48 hours for colonies to appear. Hazardous to
laboratory personnel and should be sent to laboratories with
appropriate expertise when possible.
Increases sensitivity for detection of Y. pestis over cytology
alone, but false negatives can still occur. Recent antibiotic
treatment may cause false-negative results. Must be per-
formed by laboratories with expertise in plague diagnosis.
Acute and convalescent serology is required for diagnosis of acute
infection, because initial results may be negative, and positive
results may reflect previous exposure rather than active infection.
534 SECTION 2  Bacterial Diseases
FIGURE 55-4  Lymph node aspirate cytology from a cat with bubonic plague. Bipolar
rods are scattered throughout the field. (From Nelson RW, Couto CG, eds. Small Animal
Internal Medicine, 4 ed. St. Louis, MO: Mosby; 2009.)
Cytologic Demonstration of Bacteria
Y. pestis is usually found in large numbers in tissues. If plague is
suspected, aspirates of buboes or other affected tissues should be
collected and smears prepared in isolation while wearing protec-
tive clothing, including eye protection and properly fitted high-
density face masks (see Chapter 11). The smears should be heat
fixed or immediately immersed in fixative solution to inactivate
any organisms present. Cytologic examination shows massive
numbers of neutrophils and a monomorphic population of coc-
cobacilli with bipolar staining (Figure 55-4). This is in contrast to
cat bite abscesses, where a mixture of bacteria is usually present.
Direct Immunofluorescent Antibody and Antigen ELISA Assays
Heat-fixed slides prepared from lymph node aspirates can be
submitted to approved testing facilities for application of immu-
nofluorescent antibody against the F1 envelope antigen. Local
public health authorities should be contacted for information on
recommended testing facilities if immunofluorescent antibody
(IFA) testing is required. IFA can also be applied to impression
smears from tissues obtained at necropsy. However, negative IFA
results do not rule out plague, so culture and serology should also
be considered.5 A lateral-flow dipstick assay has been described
for rapid (within 15 minutes), sensitive, and specific in-clinic
diagnosis of plague in human patients, although false negatives
may still occur.12,13 This assay detects F1 antigen in body fluids
such as sputum, lymph node aspirates, serum, and urine and has
an estimated shelf life of about 2 years at room temperature. It
has not been validated for diagnosis of plague in cats.
Isolation and Identification
Y. pestis can be grown from clinical specimens on routine media
used for isolation of organisms that belong to the Enterobacte-
riaceae. The organism grows slowly, and colonies are smaller
than those of other Enterobacteriaceae, so cultures must be held
for at least 48 hours. If plague is suspected, specimens should
never be shipped to routine veterinary diagnostic laboratories.
Instead, local public health authorities should be contacted
immediately for instructions regarding how and where to sub-
mit specimens for culture of Y. pestis.
Serology
Acute and convalescent serology is required for serologic diag-
nosis of plague in dogs and cats. The most widely used serologic
TABLE 55-2
Suggested Antimicrobial Drugs for Treatment of Plague in Cats
Dose Interval
Drug (mg/kg) Route (hours) Duration
Doxycycline 10 IV or PO* 24 10 days
Gentamicin 5-8 IV, IM, SC 24 10 days
*Oral administration should be avoided for at least the first 72 hours
of treatment to minimize exposure of the caregiver to Y. pestis. For IV
doxycycline administration, dilute in 100 mL of lactated Ringer’s saline
and administer over 2 hours; if fluid overload is a concern, consider
parenteral tetracycline or use of an aminoglycoside.
test is a passive hemagglutination inhibition (HI) assay (see
Chapter 2), which is available through public health laborato-
ries approved for plague diagnosis. Because of the extremely
short incubation period, a significant proportion of affected cats
and dogs may have negative titers when they first are brought
to the veterinarian, so convalescent serum should be submitted
at least 10 to 14 days after the initial titer. Some cats may die
before a rise in antibody titer occurs.11 A fourfold increase in
titer together with consistent clinical abnormalities confirms the
diagnosis of plague. Titers in affected cats in one study ranged
from less than 1:4 for acute specimens to 1:8192 for convales-
cent specimens.5
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays have been described for detection of Y. pestis DNA
and are an attractive means of diagnosis because of their sen-
sitivity and rapid turnaround time. In addition, the organism
can be deliberately inactivated before specimens are submitted
to the laboratory for PCR testing, which reduces the risk of
laboratory-acquired infection. Real-time PCR assays have been
validated for use on clinical specimens from humans,13 but to
date assays have not been validated for diagnosis of the disease
in cats or dogs. Real-time PCR was considerably more sensitive
for diagnosis of human plague in Africa than the lateral-flow
dipstick antigen assay or culture.
Pathologic Findings
Gross pathologic findings in cats with plague include enlarged
lymph nodes or abscesses with associated edema and hemor-
rhage, focal (up to 1 cm) lung lesions, and hemorrhages in a
variety of organs. Histopathology reveals hemorrhage, necrosis,
neutrophilic infiltrates, and often very large numbers of small
coccobacilli in tissues.
Treatment and Prognosis
Treatment of cats and dogs with plague involves supportive
care, abscess drainage, and antimicrobial drug administra-
tion (Table 55-2). Animals suspected to have plague should
be housed in isolation at least for the first 72 hours of anti-
microbial drug therapy and handled by as few individuals as
possible. Public health authorities should be notified immedi-
ately. Gowns, gloves, and suitable face protection (eye protec-
tion and a properly fited high-density surgical mask) should
be worn, and protective wear should be properly removed
and disposed of after handling affected cats. Cats and dogs
 CHAPTER 55  Yersinia pestis (Plague) and Other Yersinioses
FIGURE 55-5  Axillary bubo with ulceration and eschar formation at the site of an
infected flea bite. (Centers for Disease Control and Prevention.) (From Dennis DT, Mead
PS. Yersinia species, including plague. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell,
Douglas and Bennett’s Principles and Practice of Infectious Diseases, 7 ed, Philadelphia,
PA: Churchill Livingstone, Elsevier; 2010. )
with plague should also be treated for fleas. Abscesses should
be lanced and flushed with chlorhexidine solution. Any bio-
hazardous waste should be double bagged and disposed of
correctly. Routine disinfectants can be used to inactivate the
organism in the environment. Antimicrobials with activity
against most isolates of Y. pestis are aminoglycosides or tetra-
cyclines such as doxycycline. Trimethoprim-sulfamethoxazole
and chloramphenicol also have activity against Y. pestis. Par-
enteral aminoglycosides such as gentamicin are preferred for
seriously ill animals. Mortality rates in naturally infected cats
that are not treated with antimicrobial drugs can reach 75%,
but most (>90%) of cats treated with antimicrobial drugs
survive.5 The time between antimicrobial drug treatment and
recovery of affected cats ranged from 1 to 10 days in one study
(mean, 4.4 days).5
Immunity and Vaccination
There are no plague vaccines for dogs and cats. Although vac-
cines have been used to prevent plague in highly at-risk humans,
these did not protect against pneumonic plague. Currently there
are no vaccines approved for use in the United States. Because
Y. pestis is a potential agent of bioterrorism, efforts have been
focused on development of subunit vaccines. However, Y. pes-
tis has also been considered an unlikely agent of bioterrorism
because in general it does not survive well in the environment,
and person-to-person transmission requires close contact with a
dying person, usually on the last day of life.2,14
Prevention
Prevention of plague in dogs and cats involves housing cats
indoors in endemic areas and discouragement of predation or
exposure to/consumption of wild animal carcasses. Dogs should
not be allowed to dig around rodent burrows in endemic areas.
Flea preventatives should reduce the chance that dogs and cats
will carry infected fleas into the house. Rodent control in the
environment (e.g., removal of garbage and food scraps) should
also be performed.
535
Public Health Aspects
The clinical course of plague in humans closely resembles that
in cats (Figure 55-5). Three major pandemics of plague have
occurred in humans: Justinian plague, which occurred in the
Mediterranean basin during the sixth century Byzantine era; the
“Black Death,” which began in 1347 and spread throughout
Europe; and modern plague, which began in China in the 1860s
and was subsequently spread by ship rats to all continents inhab-
ited by humans. Currently, more than 95% of human plague
cases reported to the WHO originate from sub-Saharan Africa
and Madagascar.15 From 1970 to 2007, 415 cases were reported
in the United States, with 59 deaths.16 In endemic regions,
humans who have close contact with rodents or their canine
or feline predators are at risk. The vast majority of humans are
infected as a result of contact with rodent fleas. Human disease
has also been reported after skinning of rabbits or contact with
wild carnivores. Fewer than 10% of human plague cases result
from exposure to cats, with infection transmitted from cats to
humans after a bite, scratch, or inhalation of respiratory droplets
from cats with pneumonic plague.17-19 However, exposure to cats
often results in pneumonic plague, which has the worst prognosis
and can progress to death within as little as 24 hours after the
onset of symptoms. Occasionally, dogs or cats carry rodent fleas
into the house, which may then transmit the infection to humans.
Sleeping with a dog may also increase the risk of infection.20
Exposure to infected fleas carried into a household by a dog was
suspected in a report of two affected humans in Oregon, one of
whom slept in the same bed with the pet dog during the 2 weeks
before onset of illness.21 The dog seroconverted to Y. pestis.
Flea control and reduction of pet exposure to rodents also
serve to reduce plague in humans. Pet owners should be made
aware that pets can carry fleas (and ticks) into the house and that
flea control may reduce the chance of arthropod-­transmitted
human disease. Adoption of wild animals should be discour-
aged. In the veterinary hospital situation, plague can be pre-
vented if there is a high index of suspicion for the disease and
appropriate precautions are taken (see Treatment and Progno-
sis). Owners of pets suspected to have plague should be advised
to contact their physicians without delay. Humans with expo-
sure to affected animals are generally treated with prophylactic
doxycycline for 7 days.
Other Yersinioses
Y. pseudotuberculosis and Y. enterocolitica are enteropatho-
genic bacteria that are distributed worldwide. They are classified
into serotypes based on their O antigens, with different classi-
fication systems used for the two pathogens. Enteropathogenic
yersiniae are maintained in a variety of food animal and wild
animal reservoirs and are well known causes of food-borne febrile
gastrointestinal illness in humans. Pigs are a major reservoir for
Y. enterocolitica, and outbreaks have followed consumption of
undercooked pork products. Y. enterocolitica can also grow in
food held at refrigeration temperatures. Affected human patients
frequently exhibit right lower abdominal pain due to mesenteric
adenitis or ileitis, which can mimic appendicitis. A small pro-
portion of patients develop complications such as secondary
polyarthritis and erythema nodosum (panniculitis).16 Children
are most susceptible. Immunocompromised humans (such as
those with diabetes mellitus, malignancy, or immunosuppressive
drug therapy) can occasionally develop bacteremia with systemic
536 SECTION 2  Bacterial Diseases
spread and abscesses in a variety of different organs. The mortal-
ity rate in this situation is at least 50%.16
Dogs and cats can shed Y. enterocolitica and Y. pseudo-
tuberculosis in their feces in the absence of clinical signs.22,23
Y. enterocolitica was also isolated from 19% of 144 tonsils from
stray dogs in Ireland.24 Strains found in dogs can resemble those
implicated in human disease,25 and in several reports, dogs were
suspected as a source for human infection and disease. Y. entero-
colitica infection was associated with hepatitis in a 2-year-old
Pekingese dog from Korea26 and hepatitis and myocarditis in a
4-month-old Rottweiler.24 It has also been isolated from young
dogs with hemorrhagic diarrhea. There have been several reports
of Y. pseudotuberculosis infection in cats, with microabscess
formation in a variety of abdominal organs.27-29 A young adult
dog evaluated at the authors’ hospital for hemoptysis and lung
consolidation had pulmonary Y. pseudotuberculosis infection.
Enteropathogenic Yersinia species are usually susceptible to
doxycycline, trimethoprim-sulfamethoxazole, chloramphenicol,
aminoglycosides, and fluoroquinolones. Some organisms pro-
duce β-lactamase enzymes.
In dogs and cats, prevention of enteropathogenic yersiniosis
involves feeding properly cooked foods and preventing preda-
tion. Other measures to reduce zoonotic transmission include
hand washing after handling dogs and cats (especially before
eating), wearing gloves when contact with saliva or feces is
anticipated, and preventing dogs from licking the face or open
wounds.
CASE EXAMPLE
Refer to Chapter 56, Tularemia, for a case example.
SUGGESTED READINGS
Butler T. Plague into the 21st century. Clin Infect Dis. 2009;49:736-
742.
Eidson M, Thilsted JP, Rollag OJ. Clinical, clinicopathologic, and
pathologic features of plague in cats: 119 cases (1977-1988). J Am
Vet Med Assoc. 1991;199:1191-1197.
Wilkie M, McGivern T, Skeels M, et  al. Notes from the field: Two
cases of human plague—Oregon, 2010. Morb Mortal Wkly Rep.
2011;60:214.
REFERENCES
1. Yersin A. La peste bubonique a Hong Kong. Ann Inst Pasteur
(Paris). 1894;8:662-667.
2. Butler T. Plague into the 21st century. Clin Infect Dis. 2009;49:
736-742.
3. Eisen RJ, Borchert JN, Holmes JL, et  al. Early-phase transmis-
sion of Yersinia pestis by cat fleas (Ctenocephalides felis) and their
potential role as vectors in a plague-endemic region of Uganda. Am
J Trop Med Hyg. 2008;78:949-956.
4. Lorange EA, Race BL, Sebbane F, et al. Poor vector competence of
fleas and the evolution of hypervirulence in Yersinia pestis. J Infect
Dis. 2005;191:1907-1912.
5. Eidson M, Thilsted JP, Rollag OJ. Clinical, clinicopathologic, and
pathologic features of plague in cats: 119 cases (1977-1988). J Am
Vet Med Assoc. 1991;199:1191-1197.
6. Eisen RJ, Enscore RE, Biggerstaff BJ, et al. Human plague in the
southwestern United States, 1957-2004: spatial models of ele-
vated risk of human exposure to Yersinia pestis. J Med Entomol.
2007;44:530-537.
7. Chomel BB, Jay MT, Smith CR, et  al. Serological surveillance of
plague in dogs and cats, California, 1979-1991. Comp Immunol
Microbiol Infect Dis. 1994;17:111-123.
8. Rose LJ, Donlan R, Banerjee SN, et  al. Survival of Yersinia
pestis on environmental surfaces. Appl Environ Microbiol.
2003;69:2166-2171.
9. Eisen RJ, Petersen JM, Higgins CL, et  al. Persistence of Yer-
sinia pestis in soil under natural conditions. Emerg Infect Dis.
2008;14:941-943.
10. Orloski KA, Eidson M. Yersinia pestis infection in three dogs. J Am
Vet Med Assoc. 1995;207:316-318.
11. Gasper PW, Barnes AM, Quan TJ, et al. Plague (Yersinia pestis) in
cats: description of experimentally induced disease. J Med Ento-
mol. 1993;30:20-26.
12. Chanteau S, Rahalison L, Ralafiarisoa L, et al. Development and
testing of a rapid diagnostic test for bubonic and pneumonic
plague. Lancet. 2003;361:211-216.
13. Riehm JM, Rahalison L, Scholz HC, et  al. Detection of Yersinia
pestis using real-time PCR in patients with suspected bubonic
plague. Mol Cell Probes. 2011;25:8-12.
14. Kool JL. Risk of person-to-person transmission of pneumonic
plague. Clin Infect Dis. 2005;40:1166-1172.
15. Stenseth NC, Atshabar BB, Begon M, et al. Plague: past, present,
and future. PLoS Med. 2008;5:e3.
16. Dennis DT, Mead PS. Yersinia species, including plague. In: Man-
dell GL, Bennett JE, Dolin R, eds. Principles and Practice of Infec-
tious Diseases. 7th ed. Philadelphia, PA: Elsevier; 2010:2943-2953.
17. Doll JM, Zeitz PS, Ettestad P, et  al. Cat-transmitted fatal pneu-
monic plague in a person who traveled from Colorado to Arizona.
Am J Trop Med Hyg. 1994;51:109-114.
18. Thornton DJ, Tustin RC, Pienaar BJ, et  al. Cat bite transmis-
sion of Yersinia pestis infection to man. J S Afr Vet Assoc. 1975;
46:165-169.
19. Werner SB, Weidmer CE, Nelson BC, et al. Primary plague pneu-
monia contracted from a domestic cat at South Lake Tahoe. Calif.
JAMA. 1984;251:929-931.
20. Gould LH, Pape J, Ettestad P, et al. Dog-associated risk factors for
human plague. Zoonoses Public Health. 2008;55:448-454.
21. Wilkie M, McGivern T, Skeels M, et al. Notes from the field: Two
cases of human plague—Oregon, 2010. Morb Mortal Wkly Rep.
2011;60:214.
22. Fenwick SG, Madie P, Wilks CR. Duration of carriage and trans-
mission of Yersinia enterocolitica biotype 4, serotype 0:3 in dogs.
Epidemiol Infect. 1994;113:471-477.
23. Fukushima H, Nakamura R, Iitsuka S, et al. Prospective systematic
study of Yersinia spp. in dogs. J Clin Microbiol. 1984;19:616-622.
24. Murphy BP, Drummond N, Ringwood T, et al. First report: Yer-
sinia enterocolitica recovered from canine tonsils. Vet Microbiol.
2010;146:336-339.
25. Wang X, Cui Z, Wang H, et  al. Pathogenic strains of Yer-
sinia enterocolitica isolated from domestic dogs (Canis familia-
ris) belonging to farmers are of the same subtype as pathogenic
Y. enterocolitica strains isolated from humans and may be a source
of human infection in Jiangsu Province, China. J Clin Microbiol.
2010;48:1604-1610.
26. Byun JW, Yoon SS, Lim SK, et  al. Hepatic yersiniosis caused by
Yersinia enterocolitica 4:O3 in an adult dog. J Vet Diagn Invest.
2011;23:376-378.
27. Iannibelli F, Caruso A, Castelluccio A, et al. Yersinia pseudotuber-
culosis in a Persian cat. Vet Rec. 1991;129:103-104.
28. Spearman JG, Hunt P, Nayar PS. Yersinia pseudotuberculosis
infection in a cat. Can Vet J. 1979;20:361-364.
29. Obwolo MJ, Gruffydd-Jones TJ. Yersinia pseudotuberculosis in the
cat. Vet Rec. 1977;100:424-425.
CHAPTER 56
Tularemia
Jane E. Sykes and Bruno B. Chomel
Overview of Tularemia
First Described: 1911, Tulare County, California1
Cause: Virulent strains of Francisella tularensis, a gram-­
negative coccobacillus (family Francisellaceae)
Major Vectors: Ticks (especially Dermacentor andersoni, Der-
macentor variabilis, and Amblyomma americanum in the
eastern United States), biting flies (western United States),
mosquitos (Scandinavia)
Affected Hosts: Some 190 mammalian species, which include
rodents, rabbits, wild carnivores, domestic dogs and cats,
sheep, horses, nonhuman primates, and humans
Geographic Distribution: Northern Hemisphere between
30 degrees and 71 degrees of latitude; most disease is
reported from the United States, where virulent strains
are most prevalent
Major Clinical Signs: Fever, lethargy, inappetence, lymph-
adenopathy, subcutaneous abscesses, splenomegaly,
hepatomegaly
Differential Diagnoses: Plague, cat bite abscess, Streptococcus
spp. infections, feline infectious peritonitis, mycobacte-
rial infections, lymphoma, sepsis caused by other gram-
negative bacteria
Human Health Significance: Cats and less commonly dogs
can transmit tularemia to humans after they are exposed
to infected rodents or rabbits. Cat bites are most com-
monly implicated.
Etiologic Agent and Epidemiology
Tularemia (also known as rabbit fever, hare fever, deerfly fever,
and lemming fever) is an extremely infectious but uncommon
zoonotic disease caused by Francisella tularensis, a small (0.2
× 0.2 × 0.7 µm), nonmotile, aerobic, encapsulated gram-nega-
tive coccobacillus. The name “tularemia” comes from the first
isolation of the organism from sick rodents in Tulare County,
California, in 1911. There are four subspecies of F. tularen-
sis: tularensis, holarctica, novicida, and mediasiatica. Subspe-
cies tularensis (also known as type A) and holarctica (type B)
cause most disease in humans. Subspecies mediasiatica is only
found in Asia and the former Soviet Union. Subspecies novicida
can cause illness in severely immunosuppressed humans and is
widely studied as a model for tularemia because of its low viru-
lence to laboratory workers.
F. tularensis subsp. tularensis has the greatest degree of viru-
lence for humans and predominates in North America, whereas the
less virulent subspecies holarctica predominates in Europe. Addi-
tional genetic variation within type A and type B subpopulations
of F. tularensis correlates with geographic distribution of cases and
the severity of disease.2 Molecular subtyping has further divided
type A into subpopulations A1a, A1b, A2a, and A2b. The highest
mortality in humans, 24%, results from infections by A1b.3
Tularemia occurs primarily in the Northern Hemisphere, espe-
cially between 30 degrees and 71 degrees of latitude (Figure 56-1).
Human disease caused by a subspecies novicida–like organism
was reported from the Northern Territory in Australia in 2003.4
Even in North America, where F. tularensis subsp. tularensis
predominates, tularemia is a rare disease, and most human cases
occur in the central and north-central United States, especially
Arkansas, Missouri, Kansas, South Dakota, and Oklahoma, with
an additional focus in Massachusetts (especially Martha’s Vine-
yard) (Figure 56-2). Despite its rarity, tularemia is a reportable
disease in the United States because F. tularensis is considered a
potential agent of bioterrorism. In western Europe, tularemia is
endemic in Scandinavia but has emerged in new parts of Sweden
and is increasingly reported in humans from more southern loca-
tions, such as Germany, France, and Spain.5 Tularemia has been
absent from the United Kingdom, with the exception of travel-
related disease. It also occurs in Russia and Japan.6
Canine and feline disease due to F. tularensis has only been
reported from the United States. Young adult cats and dogs are
usually affected. As with plague, cats are more susceptible than
dogs. Cases are reported most often from Oklahoma7-9 and
Kansas,10 but also from other locations that include Montana,11
Missouri,12 Massachusetts,13 central Virginia,14 and eastern
Oregon,15 which mirrors the distribution in humans. Tularemia
is likely underdiagnosed in cats and dogs because clinical signs
FIGURE 56-1  Global distribution of tularemia (Centers for Disease Control and
Prevention).
537
538 SECTION 2  Bacterial Diseases

Ingestion of dead rabbit

by dogs or cats

Bite wound
(usually cat)

Arthropod

biting fly
A
ticks (esp. Dermacentor spp.)
Terrestrial or
aquatic
reservoirs

Inhalation

Ingestion
Cutaneous inoculation
FIGURE 56-3  Transmission cycle for Francisella tularensis.

are nonspecific. Many cats with tularemia are diagnosed with

tularemia at necropsy.7,10 The seroprevalence in pet cats from
Connecticut and New York state was 12%.16
F. tularensis is maintained in the environment by a huge
variety of terrestrial and aquatic mammals, which include rab-
Type A Human Type B Human bits, hares, ground squirrels, prairie dogs, lemmings, voles,
B Type A Animal Type B Animal beavers, muskrats, water rats, and other rodents.6 It often
causes severe disease in these species, although subclinical car-
riage may also occur. It can be transmitted by arthropod bites,
inhalation or ingestion of the organism, and direct skin contact
with infected tissues (probably when there are minute breaks
in the skin) (Figure 56-3). Inhalation of as few as 10 organisms
can cause human disease, whereas larger numbers of organ-
isms (108) must be ingested for disease to occur. Cats and dogs
become infected when they hunt rodents or lagomorphs; a his-
tory of rabbit ingestion was most often described in association
with the few reported feline and canine cases.7-9 In the western
United States (Utah, Nevada, and California), the most com-
mon arthropod vectors are biting flies, whereas ticks (especially
Dermacentor ticks and Amblyomma americanum) are the most
common vectors east of the Rocky Mountains. In Europe, Der-
macentor ticks and Ixodes ricinus are the main tick vectors;
Type A1a Human Type A1b Human Type A2 Human
mosquitos are important vectors in Scandinavia.17 F. tularensis
is well known for its ability to survive up to several months
Type A1a Animal Type A1b Animal Type A2 Animal
in fresh water sources, and survival in brackish water has also
C been demonstrated.18 Survival and replication of F. tularensis
FIGURE 56-2  Geographic distribution of tularemia in the USA. Compare this with within free-living amoebae such as Hartmannella vermiformis
the geographic distribution of plague (Figure 55-3). A, Overall distribution of human may contribute to the organism’s ability to persist in water
tularemia cases. B, Distribution of type A and type B isolates from humans and animals. sources.19 The organism can also survive for years in frozen
C, Distribution of isolates from humans and animals by molecular subtype. Note that the rabbit meat.
geographic distribution of human type A cases follows the distribution of subtype A1, the
most virulent subtype being A1b. (A and B modified from Kugeler KJ, Mead PS, Janusz AM, Clinical Features
et al. Molecular epidemiology of Francisella tularensis in the United States. Clin Infect Dis
2009;48:863-870.) Signs and Their Pathogenesis
The clinical manifestations of tularemia depend on the virulence
of the infecting strain, the route of inoculation, and host immu-
nity. Although tularemia may rapidly progress to death in cats,
protracted illness has also been described.15

F. tularensis survives in macrophages through evasion of
cellular destruction by phagosomes and inhibition of the respi-
ratory burst. Several different virulence factors of F. tularensis
have been identified, which include bacterial lipopolysaccharide
(LPS), pili, a capsule, and a series of acid phosphatase enzymes.
The endotoxic activity of the LPS of F. tularensis is 1000-fold
less potent than that of Escherichia coli.20,21 Production of acid
phosphatase enzymes may be key to the organism’s ability to
inhibit the respiratory burst and survive in macrophages.22
Many stages of the organism’s intracellular lifestyle depend on
a pathogenicity island (group of virulence genes) known as the
Francisella pathogenicity island.
For the first few days after cutaneous inoculation, F. tula-
rensis replicates locally and can produce a papule that may
then ulcerate. It then disseminates to local lymph nodes, which
may be followed by bacteremia and sepsis, with infection of
systemic, primarily reticuloendothelial tissues. The organism
invades not only macrophages but also nonphagocytic cells
such as hepatocytes and alveolar epithelial cells,23 with induc-
tion of a profound inflammatory response and necrosis. Incuba-
tion periods that range from 1 to 5 days have been suspected in
naturally occurring tularemia in dogs and cats, but longer incu-
bation periods (up to 21 days) can occur in humans. Clinical
signs typically manifest as a sudden onset of fever, lethargy, and
inappetence. Other manifestations in affected cats have included
a thin body condition, icterus, hepatomegaly, vomiting, dehy-
dration, peripheral and/or abdominal lymphadenomegaly, sple-
nomegaly, and oral and/or lingual ulceration. Hypothermia and
bradycardia can occur in moribund cats7; one cat had seizures
and was nonresponsive.14 A single cat with localized cutaneous
tularemia had a chronic (1-year duration) draining skin lesion in
the region of (but not involving) the mandibular lymph nodes,
in the absence of systemic signs.15 In the rare reports of natu-
rally affected dogs, clinical signs have included fever, anorexia,
and peripheral lymphadenopathy.8,24 One dog also had bilateral
mucoid ocular discharge and mild conjunctival hyperemia,8 and
another had tonsillitis.24
Physical Examination Findings
Physical examination in cats with tularemia may reveal lethargy,
fever (up to 105.1°F [40.5°C]) or hypothermia, a thin body con-
dition, and dehydration.7,9,11-13 Ulceration of the oral mucous
membranes or tongue may be present.7,13 Icterus may also
occur.25 Moribund cats may be hypothermic and bradycardic and
may have weak pulse quality. Mild to marked peripheral lymph-
adenopathy is present in most but not all cats. Lymphadenopathy
may be restricted to the lymph nodes of the head and neck13 or
may be generalized.7,9 Abdominal palpation may reveal hepa-
tomegaly or splenomegaly, or abdominal lymph nodes may be
palpable. Fever, lethargy, peripheral lymphadenopathy, mucoid
ocular discharge, and tonsillitis may be present in dogs.8,24
Diagnosis
Tularemia should be considered in cats and dogs with lymphade-
nopathy and fever that have a history of residence in or recent
travel to an endemic area, as well as contact with or predation
of wild mammals (especially lagomorphs). Plague is the major
differential diagnosis of zoonotic importance in the southwest-
ern United States (Table 56-1). Ultimately, the diagnosis can be
confirmed by culture, PCR, and/or serologic testing. With the
exception of PCR, access to approved laboratories that practice
CHAPTER 56  Tularemia 539
biosafety level 3 procedures is required to perform these tests
when tularemia is suspected. Local public health authorities
should be contacted to report suspicion for the disease and for
information on recommended testing facilities.
Laboratory Abnormalities
Complete Blood Count, Biochemistry Profile, and Urinalysis
Leukocyte counts in a limited number of cats with tularemia have
been normal, decreased (as low as 1300 cells/µL), or increased
with evidence of neutrophil toxicity.7,9,13,14 Moderate to marked
bandemia may be present.9,13 Thrombocytopenia is a common
finding (as low as 41,000 platelets/µL).7,9 The serum biochem-
istry panel may show hypoglycemia, azotemia, and electrolyte
abnormalities, increased liver enzyme activities, and hyperbiliru-
binemia (up to 7.6 mg/dL).7,9,14 Bilirubinuria and hemoglobin-
uria may be present.7 Mature neutrophilia, lymphopenia, and
increased serum ALP activity were present in a dog with tula-
remia.8 In human patients, abnormalities include thrombocyto-
penia, elevated serum transaminase activities, increased serum
creatine kinase activity, myoglobinuria, and pyuria.26
Cytologic Examination of Lymph Node Aspirates
Cytologic examination of lymph node aspirates in cats with
tularemia may reveal lymph node hyperplasia.9 Lymph nodes
may also contain necrotic debris and increased numbers of mac-
rophages and/or degenerate neutrophils.7,13 Because F. tularen-
sis is tiny and stains only faintly, organisms are less likely to be
visualized than those of Yersinia pestis (see Chapter 55).
Diagnostic Imaging
Plain Radiography
Findings on thoracic radiography in cats with tularemia have
not been well described; one cat had normal thoracic radio-
graphs.9 Findings on abdominal radiography have included
hepatomegaly and fluid-filled intestinal loops.9
Sonographic Findings
Abdominal ultrasound findings in cats with tularemia could
include abdominal lymphadenomegaly, splenomegaly, and/or
hepatomegaly. Abdominal lymphadenomegaly was described in
one cat.9
Microbiologic Tests
Diagnostic assays currently available for tularemia in dogs and
cats are described in Table 56-2.
Direct Detection
Heat-fixed slides can be submitted to experienced laborato-
ries for direct immunofluorescent antibody (IFA) staining for
F. tularensis. Alternatively, IFA or immunoperoxidase stains can
be applied to tissues obtained at necropsy.10 Negative results
do not rule out tularemia, but in some cases IFA may detect
the organism when culture is negative.27 Fluorescence in situ
hybridization (FISH) has also been used to detect F. tularensis
in tissues and differentiate between subspecies.28 As for plague,
lateral-flow assays have been described for rapid detection of
F. tularensis LPS antigen in clinical specimens such as urine.29
The use of these assays in dogs and cats has not been described.
Isolation and Identification
F. tularensis can be grown from clinical specimens, but it is very
fastidious, grows slowly, is hazardous to laboratory personnel,
540 SECTION 2  Bacterial Diseases

TABLE 56-1
Differences and Similarities between Plague and Tularemia in Cats and Dogs in the United States
Plague
Organism Gram-negative bacillus (Enterobacteriaceae).
­Survives relatively poorly in the environment.
Grows on routine culture media used for gram-
negative bacteria.
Major Arthropod Diverse variety of rodent and rabbit fleas
vectors
Geographic location Southwestern United States
Affected hosts Cats > dogs
Usual history Rodent contact or exposure to rodent fleas
Clinical signs Acute disease with high fever and lymphadenopathy, Acute or more chronic disease (sometimes associ-
most often of the mandibular and/or retropharyn-
geal lymph nodes. Hyperbilirubinemia and icterus
do not appear to be common in affected cats.
Lymph node aspirate Neutrophilic inflammation; monomorphic popula-
cytology tion of bipolar staining bacilli often present
Antimicrobial drugs Aminoglycosides, doxycycline, chloramphenicol,
trimethoprim-sulfamethoxazole
Zoonotic potential Exposure to cats with pneumonic plague can lead
to human infection; bites and scratches have also
been implicated. Human pneumonic plague usu-
ally ­results from exposure to infected cats and can plague. Medical attention should be sought if
progress to death within a few days. Exposed hu-
mans may require prophylaxis with doxycycline.
Tularemia
Gram-negative coccobacillus (Francisellaceae). Can
persist in the environment and water sources
for months. Fastidious growth in culture; often
requires special media and prolonged incubation.
Dermacentor spp. and Amblyomma americanum
ticks
Western, southwestern, central, and eastern United
States
Cats > dogs
Rodent and especially rabbit contact; ingestion
often reported
ated with a thin body condition) with oral ulcer-
ation, lymphadenopathy of the mandibular and/
or retropharyngeal lymph nodes, or generalized
lymphadenopathy and abdominal organomegaly.
Hyperbilirubinemia is frequent in affected cats,
and icterus has been described.
Lymphoid reactivity or neutrophilic to pyogranulo-
matous inflammation and necrosis; bacteria often
not visualized
Aminoglycosides are the treatment of choice. Doxy-
cycline, chloramphenicol, and fluoroquinolones
may be effective but may be followed by relapse.
Most often follows bites from affected cats, less of-
ten scratches. Course of disease longer and mor-
tality may be lower than occurs with pneumonic
bites or scratches occur.

TABLE 56-2
Diagnostic Assays Currently Available for Tularemia in Dogs and Cats
Assay Specimen Type Target Performance
Culture Lymph node aspirates, Francisella tularensis Recent antibiotic treatment may cause false-­
blood, tissues ­collected negative results. May require special media and
at necropsy takes at least 48 hours for colonies to appear.
Hazardous to laboratory personnel and should
be sent to laboratories with appropriate expertise
when possible.
Direct immunofluorescent Lymph node aspirates, F. tularensis antigen May be positive when culture is negative. False
antibody tissues collected at negatives can still occur. Recent antibiotic
necropsy ­treatment may cause false-negative results.
Serology Serum Antibodies to Acute and convalescent serology is required
F. tularensis for diagnosis, because initial results may
be ­negative and positive results may reflect
­previous ­exposure rather than active infection.
­Seroconversion may take up to 3 weeks.

and does not always grow on media used for routine isolation
of gram-negative bacteria such as MacConkey agar (in contrast
to Y. pestis). The use of cysteine-supplemented media (such as
cysteine heart agar) and incubation in a CO2-supplemented
environment enhances recovery of the organism. Growth gener-
ally occurs 48 to 72 hours after inoculation, but cultures must
be held for 7 to 10 days. Overgrowth of contaminating bacteria
can occur unless the media is supplemented with antibiotics that
inactivate these organisms. F. tularensis may grow from blood
specimens after inoculation of routine blood culture media. The
organism is identified on the basis of biochemical testing with
or without the use of molecular methods such as 23S rRNA
gene PCR sequencing (which allow subspecies determination).
Whole-cell matrix-assisted laser desorption ionization–time of
flight (MALDI-TOF) mass spectrometry, which measures the
mass of peptides and small proteins from whole cells, has also
been used for identification of F. tularensis isolates to the sub-
species level.30
Serology
As for plague, acute, and convalescent serology, with a fourfold
increase in titer, is required for serodiagnosis of tularemia in
affected dogs and cats and is less hazardous to human health
than culture. Tube agglutination and microagglutination assays
are the standard methods for serodiagnosis, but ELISA assays
have also been developed. Because of the extremely short incu-
bation period, affected cats and dogs may have negative titers
when they first are brought to the veterinarian, so convalescent
serum should be submitted at least 2 to 3 weeks after the initial
titer if the animal survives. Some cats die from the disease before
a rise in antibody titer occurs. In addition, high titers may per-
sist after recovery from infection, so a single positive titer is not
diagnostic for tularemia.
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays that detect F. tularensis DNA represent an attractive
means of diagnosis because of their sensitivity, rapid turnaround
time, low risk to laboratory personnel, and the ability of some
assays to differentiate between subspecies and genotypes of F. tula-
rensis.31 A PCR assay was used to detect F. tularensis in tissues at
necropsy in a cat.12 A number of real-time PCR assays have been
used to detect F. tularensis in human clinical specimens. Despite
the sensitivity of PCR assays, false negatives can still occur. Real-
time PCR assays for F. tularensis are offered on a commercial basis
by some veterinary diagnostic laboratories in the United States.
Pathologic Findings
Gross pathologic findings in cats with tularemia include icterus,
oral ulceration, lymphadenomegaly, splenomegaly, and hepa-
tomegaly. Multifocal to coalescing irregular yellow, gray, or
white foci (usually 1 to 5 mm in diameter) are present in these
organs, and sometimes other organs such as the lung and myo-
cardium.7,12,13 When examined histopathologically, the nodules
represent well-circumscribed but non-encapsulated masses of
caseous necrosis, with abundant cellular debris, surrounded
by infiltrates of degenerate neutrophils, macrophages, and/or
fibroblasts. Lesions are centered on the splenic white pulp and
cortical lymphoid follicles within lymph nodes. Necrosis and
hemorrhage of lymphoid follicles within the gastrointestinal
tract may be present.7,11 Organisms may be discernible with sil-
ver staining as tiny coccobacilli11 or can be demonstrated with
immunoperoxidase or immunofluorescence methods.10,27
CHAPTER 56  Tularemia 541
TABLE 56-3
Suggested Antimicrobial Drugs for Treatment of Tularemia in
Cats*
Dose Interval
Drug (mg/kg) Route (hours) Duration
Gentamicin 5-8 IV, IM, SC 24 14 days
Doxycycline 5 PO† 12 2-3 weeks
Marbofloxacin 2.75-5.5 PO 24 2-3 weeks
*The same antimicrobial drugs could be used to treat dogs; doses shown
are for cats.
†Parenteral aminoglycosides are preferred for at least the first 72 hours
of treatment to minimize exposure of the caregiver to F. tularensis dur-
ing treatment.
Treatment and Prognosis
Treatment of cats (and, although less commonly affected, dogs)
with tularemia involves supportive care and antimicrobial drug
administration (Table 56-3). As for plague, animals suspected to
have tularemia should be housed in isolation at least for the first
72 hours of antimicrobial drug therapy and handled by as few
individuals as possible. Public health authorities should be noti-
fied. Gowns, gloves, and suitable face protection (eye protec-
tion and a properly fitted high density surgical mask) should be
worn, and protective wear should be properly removed and dis-
posed of after handling affected cats. Any biohazardous waste
should be double bagged and disposed of correctly. Routine dis-
infectants should be used to inactivate the organism in the envi-
ronment. The drug of choice for initial treatment of tularemia
is a parenteral aminoglycoside such as gentamicin. Doxycycline
can also be used but is associated with a higher relapse rate in
humans than occurs with aminoglycosides.26 Fluoroquinolones
are active against F. tularensis in  vitro, but like tetracyclines,
they may be associated with relapse in vivo.32 Extension of the
course of treatment from 2 weeks to 3 weeks when fluoroquino-
lones or doxycycline is used has been effective for treatment of
human tularemia.25 Other drugs with in vitro activity against F.
tularensis include chloramphenicol and erythromycin.33
Immunity and Vaccination
Natural infection by F. tularensis is followed by protection
against reinfection. Because F. tularensis is facultatively intracel-
lular, cell-mediated immune responses are required for patho-
gen clearance, although humoral immunity is also important.23
As a result, inactivated vaccines have not been effective for pre-
vention of tularemia, because they primarily induce humoral
immunity. An attenuated live vaccine that contained F. tular-
ensis subsp. holarctica (live vaccine strain, or LVS) has been
available for prevention of serious disease in people at high risk
of exposure. However, this remains unlicensed because of con-
cerns regarding efficacy and the route of administration, which
is by scarification.34 Since then there has been strong interest
in vaccine development for prevention of human tularemia,
because of concerns that the organism will be used as a weapon
of bioterrorism.23 Vaccines are not likely to be available for
dogs and cats given the rarity of disease in these species and the
minor role that they play in transmission of disease to humans.
542 SECTION 2  Bacterial Diseases
Prevention
Prevention of tularemia in dogs and cats involves housing cats
indoors and discouragement of predation or exposure to dead
wild rodents and rabbits. Use of tick preventatives also reduces
the chance that dogs and cats will carry infected ticks into the
house. Rodent control in the environment (e.g., removal of
­garbage) should also be performed.
Public Health Aspects
Humans are most commonly infected by F. tularensis after
arthropod bites or handling infected wild animals. In the United
States, disease is most commonly reported in late spring and
summer. Occupations associated with an increased risk of tula-
remia include farming, hunting, landscaping, meat handling,
working with sheep, veterinary practice, and laboratory work.
Severe illness is most often reported in young children (5 to
9 years) and the aged (>75 years). Men are more likely to be
affected than women.26
Several overlapping forms of disease have been described
in humans, the development of which depends on the route of
inoculation and the virulence of the infecting strain: ulceroglan-
dular, glandular, oculoglandular, oropharyngeal, typhoidal, and
pneumonic.26 Ulceroglandular tularemia results from cutaneous
inoculation, oropharyngeal disease follows ingestion and pneu-
monic tularemia can result from aerosol inhalation. Pneumonic
tularemia has been described in people after activities such as
lawn mowing or brush cutting in tick-infested areas. Infection
of humans from dogs and cats can occur after these animals
carry F. tularensis in their mouths after ingestion of wild prey.
Human-to-human transmission does not occur.
Clinical signs in humans consist of acute onset of a flu-like
illness, with fever, chills, and malaise. Sore throat, abdomi-
nal pain, vomiting, diarrhea, lymphadenopathy, and a cough
may also occur. Infection with less virulent strains may result
in transient flu-like illness followed by recovery. Without treat-
ment, clinical signs can persist for several months, with chronic
lymphadenopathy, weight loss, and debilitation.26 Disease in
chronically affected humans can resemble cat scratch disease.
Ulceroglandular tularemia is the most common form of tula-
remia seen in humans. It consists of a red and painful papule
at the site of skin infection, with associated lymphadenopathy
(Figure 56-4). The papule then undergoes necrosis, forming
an ulcer, which can take weeks to heal. In some individuals,
lymph nodes may suppurate and become fluctuant. Multiple
lesions can occur on the hands when infected animals have
been handled, whereas tick bites typically result in single lesions
elsewhere on the body.26 Only lymphadenopathy may be pres-
ent if the ulcer heals before the time of evaluation (glandular
tularemia). Oculoglandular tularemia occurs when organisms
gain access through the conjunctiva and is characterized by con-
junctivitis, conjunctival ulceration, lacrimation, and associated
lymphadenopathy.26 Oropharyngeal tularemia is characterized
FIGURE 56-4  Papule undergoing central necrosis with desquamation on the thigh
of a middle-aged man infected with Francisella tularensis. (Courtesy of Dr. Joseph A. Boc-
chini, Louisiana State University Health Sciences Center, Shreveport, LA. In: Mandell GL,
Bennett JE, Dolin R, eds. Principles and Practice of Infectious Diseases, 7th ed, Philadelphia,
PA: Churchill Livingstone; 2010:2927-2937.)
by fever and severe throat pain; tonsillitis, oral ulceration, and
associated lymphadenopathy may be present, which resembles
the disease described in dogs and cats (which usually acquire
infection via ingestion). Oropharyngeal tularemia tends to
occur in children who ingest contaminated food or water and
can resemble “strep throat.”26 Typhoidal tularemia is a febrile
illness in the absence of prominent lymphadenopathy and may
be associated with sore throat, abdominal pain, diarrhea, vom-
iting, icterus, and, with more chronic illness, hepatomegaly and
splenomegaly. Pneumonic tularemia is the most severe form
and results from inhalation of the organism or hematogenous
spread of the organism to the lung, with clinical signs such as
cough, chest pain, and hemoptysis. Thoracic radiographs can
reveal lobar infiltrates, hilar lymphadenopathy, pleural effusion,
and miliary infiltrates.26 Changes resemble those seen in other
chronic pneumonias, such as tuberculosis or fungal disease.
Although not the most common route of infection for humans,
cat-bite–associated tularemia has been described (including
among veterinary staff), and cats that live in rural endemic areas
are important vectors of the most virulent strain of F. tular-
ensis, type A1b.25 Disease in humans can mimic Pasteurella
multocida cellulitis, the most common bacterial infection after
a cat bite, but treatment with amoxicillin or clavulanic acid–
amoxicillin is ineffective. Less commonly, human disease fol-
lows cat scratches or casual contact with cats.27 In some cases,
cats had recent contact with or ingested wild rabbits or rodents.
Both apparently healthy and sick cats have been involved. Aero-
sol exposure to organisms on the coats of dogs (such as occurs
when dogs shake their coats after contact with infected rabbits)
has rarely been suspected in clusters of human disease.35-37 In
summary, tularemia should especially be considered in humans
who develop illness in endemic areas within 3 weeks after a bite
from a cat that is a stray or that has had a recent history of wild
rabbit or rodent contact.

CASE EXAMPLE
Signalment: “Sam,” a 5-year-old male neutered domestic
shorthair cat from Woodland in northern California
History: Sam was brought to the University of California,
Davis, veterinary emergency service for evaluation of fever
and inappetence. The owner first noticed he was ill 4 days
previously, when he regurgitated a small amount of pink
fluid about 1 hour after eating. The following day he was
not eating and lethargic, and his water consumption was
reduced. On day 3, he was taken to a local veterinary clinic
where he was found to be febrile (105°F [40.5°C]), icteric, and
had a mass on the left side of his neck. He was treated with
enrofloxacin (4.5 mg/kg SC, once), ketoprofen (1.9 mg/kg
SC), and clavulanic acid–amoxicillin (12 mg/kg PO q12h).
The results of a CBC showed a mild neutrophilia (13,083
cells/µL; reference range, 2500-12,500 cells/µL) with slightly
toxic neutrophils and a mild lymphopenia (1176 cells/µL;
reference range, 1500-7000 cells/µL). A biochemistry panel
showed hyponatremia (133 mEq/L; reference range, 147-
156 mEq/L), hypochloremia (99 mEq/L; reference range,
111-125 mEq/L), hypokalemia (3.4 mEq/L; reference range,
3.9-5.3 mEq/L), hyperglycemia (214 mg/dL; reference range,
70-150 mg/dL), hyperbilirubinemia (3.7 mg/dL; reference
range, 0-0.4 mg/dL), increased serum CK activity (1858 U/L;
reference range, 64-440 U/L), and a mild metabolic acidosis
(bicarbonate 12 mEq/L; reference range, 13-25 mEq/L).
ELISA serology for FeLV antigen and FIV antibody and IFA
serology for FCoV antibody were negative. On receipt of the
laboratory test results, the local veterinarian recommended
referral.
Sam had been fed commercial dry and canned cat food and
was treated monthly with fipronil. He had been acquired at
6 weeks of age, at which time he was neutered. He had been
vaccinated annually by the owner for feline respiratory vi-
ruses and feline panleukopenia virus, and was vaccinated for
rabies for the first time 4 months earlier. He lived with three
other cats, three dogs, and two rabbits, and the owner had
brought home a wild mouse from the Lake Tahoe (Sierras) re-
gion several weeks before Sam became ill. The rabbits were
acquired by the owner 6 months ago, were kept indoors, and
did not receive flea control. The owner fostered cats and
occasionally dogs for the local animal shelter and cared for
a colony of feral cats, which did not interact with the house-
hold cats. The household cats lived harmoniously indoors.
Physical Examination:
Body Weight: 5.4 kg
General: Lethargic but responsive. Resented handling. T =
105.9°F [41.1°C], RR = 60 breaths/min, HR = 176 beats/min,
CRT <2 s.
Integument: Mild scaling was present throughout the haircoat,
but no ectoparasites were visible.
Eyes, Ears, Nose, and Throat: No clinically significant
abnormalities were present.
Musculoskeletal: Body condition score 6/9. There was a 2 × 2
× 6 cm firm subcutaneous mass alongside the trachea on
the left side.
Cardiovascular: There were no murmurs or arrhythmias
detected on cardiac auscultation. Pulses were strong and
synchronous.
CHAPTER 56  Tularemia 543
Respiratory: Tachypnea was present with a shallow respiratory
pattern. Normal bronchovesicular sounds were present in all
lung fields.
Abdominal Palpation: No clinically significant abnormalities
were identified. The urinary bladder was palpable but
small.
Lymph Nodes: The left mandibular lymph node was enlarged
at 2 cm. All other lymph nodes appeared to be within normal
limits.
Laboratory Findings:
CBC:
HCT 20.3% (30%-50%)
MCV 42.9 fL (42-53 fL)
MCHC 33.0 g/dL (30-33.5 g/dL)
Reticulocytes 9500 cells/µL (7000-60,000 cells/µL)
WBC 23,120 cells/µL (4500-14,000 cells/µL)
Neutrophils 18,958 cells/µL (2000-9000 cells/µL)
Band neutrophils 3699 cells/µL
Lymphocytes 231 cells/µL (1000-7000 cells/µL)
Monocytes 231 cells/µL (50-600 cells/µL)
Eosinophils 0 cells/µL (150-1100 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL)
Platelets 102,000/µL (180,000-500,000 platelets/µL).
Serum Chemistry Profile:
Anion gap 18 mmol/L (13-27 mmol/L)
Sodium 143 mmol/L (151-158 mmol/L)
Potassium 3.2 mmol/L (3.6-4.9 mmol/L)
Chloride 110 mmol/L (117-126 mmol/L)
Bicarbonate 18 mmol/L (15-21 mmol/L)
Phosphorus 3.0 mg/dL (3.2-6.3 mg/dL)
Calcium 8.8 mg/dL (9.0-10.9 mg/dL)
BUN 15 mg/dL (18-33 mg/dL)
Creatinine 0.8 mg/dL (1.1-2.2 mg/dL)
Glucose 151 mg/dL (63-118 mg/dL)
Total protein 6.6 g/dL (6.6-8.4 g/dL)
Albumin 2.5 g/dL (2.2-4.6 g/dL)
Globulin 4.1 g/dL (2.8-5.4 g/dL)
ALT 30 U/L (27-101 U/L)
AST 39 U/L (17-58 U/L)
ALP 5 U/L (14-71 U/L)
GGT < 3 U/L (0-4 U/L)
Cholesterol 134 mg/dL (89-258 mg/dL)
Total bilirubin 2.1 mg/dL (0-0.2 mg/dL)
Magnesium 2.0 mg/dL (1.5-2.5 mg/dL)
Creatine kinase 586 U/L (73-260 U/L).
Urinalysis: SGr 1.014; pH 8.0, trace protein (SSA), 1+ bilirubin,
2+ hemoprotein, 2+ glucose, rare WBC/HPF, 7-12 RBC/HPF,
few lipid droplets.
Imaging Findings:
Thoracic Radiographs and Abdominal Ultrasound: No
clinically significant abnormalities were present.
Cervical Ultrasound: There was moderate to severe
enlargement of the retropharyngeal lymph nodes bilaterally
and of the left mandibular salivary gland.
Microbiologic Testing: Cytologic examination of lymph
node aspirates (mandibular and retropharyngeal): A
slightly bloody background was admixed with markedly
increased numbers of variably degenerate neutrophils.
Low numbers of lymphocytes and scattered macrophages
are also observed. Interpretation: marked suppurative
inflammation.
Continued
544 SECTION 2  Bacterial Diseases
Comments: Although no intracellular bacteria were seen, the
number of neutrophils and their variably degenerate nature
suggested sepsis as the primary differential diagnosis.
Aerobic and anaerobic bacterial culture and susceptibility
(lymph node aspirate): No growth.
PCR for Francisella tularensis DNA (lymph node aspirate): Negative.
Diagnosis: Severe suppurative lymphadenitis and fever;
suspect plague or tularemia.
Treatment: Sam was transferred to isolation, during which
time he bit a clinician, and treated with intravenous fluids
(lactated Ringer’s solution and 20 mEq/L KCl at 15 mL/h IV)
and antimicrobial drugs (ampicillin, 20 mg/kg IV q8h and
gentamicin, 5 mg/kg IV q24h). Public health officials were
contacted. The cat’s rectal temperature initially increased
to 107°F (41.6°C); dark brown diarrhea was also noted. On
the second day of hospitalization, Sam’s rectal temperature
was 105°F (40.5°C), and by day 3 it had normalized and
Sam began to eat. It increased again to 103.9°F (40.0°C) the
following day, and the right mandibular and retropharyngeal
lymph nodes became palpably enlarged. Subsequently,
the cat’s rectal temperature normalized, laboratory
abnormalities improved, and the lymph nodes decreased
in size and were soft on palpation. The cat was discharged
with instructions to continue treatment with oral amoxicillin
and subcutaneous gentamicin for 7 days, with a recheck
planned at that time for an examination and convalescent Y.
pestis and F. tularensis serology. The owner did not bring Sam
back for a recheck examination, but a follow-up telephone
conversation indicated that Sam had recovered completely.
SUGGESTED READINGS
Kugeler KJ, Mead PS, Janusz AM, et  al. Molecular epidemiology of
Francisella tularensis in the United States. Clin Infect Dis. 2009;48:
863-870.
Spagnoli ST, Kuroki K, Schommer SK, et al. Pathology in practice. Fran-
cisella tularensis. J Am Vet Med Assoc. 2011;238:1271-1273.
Weinberg AN, Branda JA. Case records of the Massachusetts General
Hospital. Case 31-2010. A 29-year-old woman with fever after a cat
bite. N Engl J Med. 2010;363:1560-1568.
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miology. Future Microbiol. 2010;5:649-661.
3. Kugeler KJ, Mead PS, Janusz AM, et  al. Molecular Epidemiol-
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4. Whipp MJ, Davis JM, Lum G, et al. Characterization of a novicida-
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Comments: A diagnosis of plague or tularemia was never
confirmed in this cat, although strongly suspected based
on the history of wild rodent and rabbit exposure (with
the rodent adopted from a plague endemic region), and
the clinical abnormalities that were present. The findings
of hyperbilirubinemia, thrombocytopenia, and increased
serum CK activity were perhaps more suggestive of
tularemia than plague, although hyperbilirubinemia and
thrombocytopenia can occur in human plague. The history
of antibiotic treatment may have contributed to the negative
cytology, culture, and PCR results. Culture was requested on
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were initially overlooked as likely diagnoses. Once these
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specimens out to a public health laboratory for additional
testing, but this was complicated by concerns that related to
the potentially hazardous nature of the specimens. Handling
of the cat was limited to as few individuals as possible and
was difficult because the cat was aggressive. The clinician
who was bitten by the cat immediately saw her physician,
who prescribed doxycycline for 7 days. Rabies quarantine
was also instituted because the cat was incompletely
vaccinated for rabies. The owner was educated about the
possibility of plague or tularemia and the implications for
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animals received flea prevention, and there was significant
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9. Woods JP, Crystal MA, Morton RJ, et al. Tularemia in two cats.
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11. Rhyan JC, Gahagan T, Fales WH. Tularemia in a cat. J Vet Diagn
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12. Spagnoli ST, Kuroki K, Schommer SK, et al. Pathology in practice.
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13. Gliatto JM, Rae JF, McDonough PL, et al. Feline tularemia on Nan-
tucket Island. Massachusetts. J Vet Diagn Invest. 1994;6:102-105.
14. Inzana TJ, Glindemann GE, Snider G, et al. Characterization of a
wild-type strain of Francisella tularensis isolated from a cat. J Vet
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15. Valentine BA, DeBey BM, Sonn RJ, et  al. Localized cutaneous
infection with Francisella tularensis resembling ulceroglandular
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16. Magnarelli L, Levy S, Koski R. Detection of antibodies to Fran-
cisella tularensis in cats. Res Vet Sci. 2007;82:22-26.
17. Lundstrom JO, Andersson AC, Backman S, et  al. Transstadial
transmission of Francisella tularensis holarctica in mosquitoes.
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18. Berrada ZL, Telford Iii SR. Survival of Francisella tularensis Type
A in brackish-water. Arch Microbiol. 2011;193:223-226.
19. Santic M, Ozanic M, Semic V, et al. Intra-vacuolar proliferation of
F. novicida within H. vermiformis. Front Microbiol. 2011;2:78.
20. Ancuta P, Pedron T, Girard R, et  al. Inability of the Fran-
cisella tularensis lipopolysaccharide to mimic or to antagonize
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21. Gunn JS, Ernst RK. The structure and function of Francisella lipo-
polysaccharide. Ann N Y Acad Sci. 2007;1105:202-218.

22. Dai S, Mohapatra NP, Schlesinger LS, et al. The acid phosphatase
AcpA is secreted in vitro and in macrophages by Francisella spp.
Infect Immun. 2012;80:1088-1097.
23. Barry EM, Cole LE, Santiago AE. Vaccines against tularemia. Hum
Vaccin. 2009;5:832-838.
24. Gustafson BW, DeBowes LJ. Tularemia in a dog. J Am Anim Hosp
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25. Weinberg AN, Branda JA. Case records of the Massachusetts Gen-
eral Hospital. Case 31-2010. A 29-year-old woman with fever after
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26. Penn RL. Francisella tularensis (tularemia). In: Mandell GL, Ben-
nett JE, Dolin R, eds. Principles and Practice of Infectious Dis-
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27. Capellan J, Fong IW. Tularemia from a cat bite: case report
and review of feline-associated tularemia. Clin Infect Dis.
1993;16:472-475.
28. Splettstoesser WD, Seibold E, Zeman E, et al. Rapid differentiation
of Francisella species and subspecies by fluorescent in situ hybrid-
ization targeting the 23S rRNA. BMC Microbiol. 2010;10:72.
29. Grunow R, Splettstoesser W, McDonald S, et  al. Detection of
Francisella tularensis in biological specimens using a capture
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CHAPTER 56  Tularemia 545
30. Seibold E, Bogumil R, Vorderwulbecke S, et al. Optimized appli-
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31. Molins CR, Carlson JK, Coombs J, et  al. Identification of Fran-
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32. Steward J, Piercy T, Lever MS, et  al. Treatment of murine
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33. Urich SK, Petersen JM. In  vitro susceptibility of isolates of Fran-
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34. Conlan JW, Oyston PC. Vaccines against Francisella tularensis.
Ann N Y Acad Sci. 2007;1105:325-350.
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CHAPTER 57
Bite and Scratch Wound Infections
Jane E. Sykes
Overview of Bacterial Bite Wound Infections
Causes: A variety of aerobic and anaerobic bacteria that include
mycoplasmas and mycobacterial species. Pasteurella spp.
are commonly isolated.
Geographic Distribution: Worldwide
Major Clinical Signs: Fever, lethargy, inappetence, hyperes-
thesia, puncture wounds, cutaneous swellings, lacera-
tions, tissue avulsions, and signs that relate to damage
and/or infection of other structures (such as the thorax,
abdomen, and brain and spinal cord)
Differential Diagnoses: Other traumatic injuries, cutaneous neo-
plasia, sterile nodular panniculitis (dogs), plague, tularemia
Human Health Significance: Cat and dog bite wound infec-
tions are common in humans and may be life threatening
in the immunocompromised. They also can be associated
with transmission of potentially serious bloodborne bac-
terial diseases such as bartonellosis and tularemia.
Etiology and Epidemiology
Bite wounds are one of the most common reasons that dogs
or cats are brought to veterinary hospitals for care. Despite
this, there is surprisingly little information available on their
epidemiology in comparison to the plethora of publications on
animal bite wounds in people (see Public Health Aspects). Bite
wounds often result in bacterial infection, but can also result
in systemic viral infection (especially in the case of bitten cats),
and less often, fungal or bloodborne protozoal infections such
as babesiosis (Table 57-1).
Bacterial pathogens that cause bite wound infections are
usually members of the normal oral cavity flora of the biting
animal species, but organisms in the environment can also
contaminate bite or scratch wounds (e.g., Clostridium tet-
ani, Mycobacterium spp., or Nocardia spp.). Bite wounds are
instantly contaminated with millions of bacterial species from
the oral cavity, but only a small proportion of these species pos-
sess virulence properties that allow them to proliferate within
the wound and cause disease. The degree to which proliferation
occurs also depends on host factors such as underlying immu-
nocompromise. The bacterial species that cause bite wound
infections in dogs and cats differ from those that cause dog
or cat bite wound infections in human patients, so data from
human studies cannot be extrapolated to dog or cat bite wound
infections.
546
Dogs
Bite wounds in dogs most often result from aggressive dog-dog
interactions. The dog breeds affected depend on regional variation
in dog breed popularity. Younger adult, male dogs and especially
intact male dogs appear to be predisposed, but studies that compare
affected dogs to a control (or “background”) population have not
been reported.1-3 Bite wounds in dogs can also result from the bites
of other domestic and wild animal species, but the epidemiology
of infections that result from these wounds is not well described.
Approximately 20% of dog-dog bite wounds become
infected, as opposed to contaminated. The likelihood that a
wound becomes infected increases with the time lag between the
aggressive interaction and when veterinary attention is sought.2,3
Infection develops 8 to 24 hours after the bite occurs and is less
likely to be develop if the wound is limited to the dermis.3 The
distribution of bacterial species involved varies from one study
to another and may be influenced by whether infected or only
contaminated wounds were evaluated. The most common bac-
terial species isolated from dog-dog bite wounds are Staphylo-
coccus pseudintermedius, Enterococcus spp., Pasteurella spp.,
streptococci, and Escherichia coli.1-3 Staphylococcus aureus and
Capnocytophaga canimorsus, which are important pathogens
in humans that are bitten by dogs, are rarely isolated from bit-
ten dogs,2,3 although the prevalence of C. canimorsus is proba-
bly underestimated because it is very difficult to grow in culture.
In the author’s hospital, Pasteurella spp. accounted for 17% of
41 bacterial species cultured from 33 dog bite wounds, followed
by a variety of staphylococci (some methicillin-­resistant) (15%),
E. coli (12%), Enterococcus spp. (7%), and Bacteroides spp.
(7%). Other organisms were Peptostreptococcus spp., Clostrid-
ium spp., and Pseudomonas aeruginosa (each 5%); and Strepto-
coccus viridans, Actinomyces spp., Acinetobacter spp., Serratia
marcescens, Klebsiella pneumoniae, Myroides spp., Prevotella
spp., Fusobacterium spp., Aeromonas spp., Mycobacterium
smegmatis, and Mycoplasma spp. (one isolate each or 2%).
Obligate anaerobes were isolated from 5 dogs (15%), and in all
5 of these dogs, multiple bacterial species were isolated.
Cats
Most bite wounds in cats result from aggressive interactions with
other cats, but nonfatal dog bite wounds can also occur. Occasion-
ally, cats are bitten by a variety of other small wildlife species that
vary geographically based on the local fauna present. In contrast
to dogs, which often crush and tear tissues with their teeth, cats
deliver deep puncture wounds that create an environment where
obligate and facultative anaerobes flourish. Thus, anaerobic bac-
terial infections are more prevalent in cat bite abscesses than in
dog bite wounds, and accordingly, polymicrobial infections are
 CHAPTER 57  Bite and Scratch Wound Infections 547

TABLE 57-1
Pathogens That Cause Local or Systemic Infections in Dogs and Cats as a Consequence of Bite or Scratch Wounds from the Same or
Another Animal Species
Organism Class Dogs Cats
Viruses Rabies Rabies
FeLV
FIV
Mycoplasmas Hemotropic mycoplasmas? Nonhemotropic Mycoplasma spp.
Hemotropic mycoplasmas?
Gram-negative Pasteurella spp. Pasteurella spp.
aerobes* Escherichia coli Escherichia coli
Pseudomonas aeruginosa
Acinetobacter spp.
Proteus spp.
Enterobacter spp.
Serratia marcescens
Aeromonas spp.
Capnocytophaga canimorsus
Gram-positive Staphylococcus spp., especially S. pseudintermedius Streptococcus spp.
aerobes* Streptococcus spp. Enterococcus spp.
Enterococcus spp. Actinomyces spp.
Actinomyces spp. Nocardia spp.
Corynebacterium spp. Lactobacillus spp.
Corynebacterium spp.
Anaerobes Bacteroides spp. Bacteroides spp.
Clostridium spp. Prevotella spp.
Porphyromonas spp. Porphyromonas spp.
Fusobacterium spp. Fusobacterium spp.
Peptostreptococcus spp. Clostridium spp.
Propionibacterium spp. Propionibacterium spp.
Prevotella spp.
Eubacterium spp.
Mycobacteria Tuberculous mycobacteria (e.g., M. bovis in the Tuberculous mycobacteria (e.g., M. microti in the
United Kingdom) United Kingdom)
Rapidly growing mycobacteria Rapidly growing mycobacteria (e.g., M. fortuitum)
Lepromatous mycobacteria (e.g., M. lepraemurium)
Fungi Sporothrix schenckii Sporothrix schenckii
Opportunistic molds (e.g., Paecilomyces spp.), dematia-
ceous molds
Protozoa Babesia gibsoni None known
Babesia conradae?

*The term ‘aerobe’ refers to facultative anaerobes and obligate aerobes.

present more often in closed cat bite abscesses than in dog bite
wounds. In a study of 36 closed cat bite abscesses, 168 bacte-
rial strains were isolated, of which 72% were obligate anaerobes
and 28% were facultative anaerobes.4 The most prevalent anaer-
obes isolated from cat bite abscesses include Porphyromonas,
Bacteroides, Prevotella, Peptostreptococcus, and Fusobacterium.
Pasteurella multocida, a commensal of the oral cavity of virtu-
ally all cats, is the most common facultative anaerobe present.4-6
Porphyromonas spp. appear to be particularly prevalent; in one
study of 15 abscesses in Australian cats, they accounted for 92%
to 99% of all the facultative and obligate anaerobes present.7
Less often Actinomyces, β-hemolytic streptococci, lactobacilli,
and Propionibacterium spp. have been isolated.
Clinical Features
Dogs
Among dogs, dog bite wounds consist of abrasions, lacerations,
avulsions (i.e., skin flaps), crushing injuries, and deep puncture
wounds (Figure 57-1). Abscesses can also develop. Dog bite
wounds may also penetrate body cavities and cause pneumotho-
rax or damage the esophagus, vertebral column, or gastrointesti-
nal tract. Pyothorax or bacterial peritonitis can also occur. Most
dogs have between 1 and 5 wounds. Rarely, more than 10 wounds
are present.1-3 The majority of bite wounds occur cranial to the
diaphragm, especially on the head and neck (Figure 57-2). The
location of the bite wounds also depends on the size of the bitten
548 SECTION 2  Bacterial Diseases

A B

C
FIGURE 57-1  Dog bite wounds. A, Multiple puncture wounds, abrasions and lacerations to the abdomen of a 10-year-old female spayed mixed breed dog. A portion of the jejunum
had perforated and had herniated into the subcutaneous tissue. The dog developed cardiac arrest after 2 days of aggressive treatment that included surgery and was euthanized. B, Five-
year-old male neutered Pomeranian dog with a bite wound to the lateral cervical region from a pit-bull terrier dog that resulted in tissue avulsion. C, Inguinal region of a 6-year-old intact
male German shepherd dog with severe bite wounds and tissue avulsion that resulted in extensive exposure of muscle and bone. The dog had been attacked by four other dogs. The dog
survived with aggressive medical treatment and surgery that included amputation of the right pelvic limb. (Courtesy of the University of California, Davis Veterinary Emergency and Critical
Care Service.)

FIGURE 57-2  Common anatomic distribution of dog-to-dog bite and fight wounds.
 CHAPTER 57  Bite and Scratch Wound Infections 549

dog; large-breed dogs are more likely to have wounds on the neck
and face, but small-breed dogs often have wounds on their dor-
sum. Injury to underlying tissue is frequently dramatically more
severe than is apparent on the surface. Classification systems have
been used to describe the severity of dog-dog bite wounds based
on the type of injury (laceration vs. puncture wound) and the
presence or absence of dead space or abscessation.1-3 Abscessa-
tion is rarely associated with dog bite wounds as compared with
cat bite wounds.3 The presence of pus, fever, erythema, subcuta-
neous emphysema, and/or a foul odor suggests that infection has
occurred. Uncommonly, hematogenous spread of bacteria leads
to signs of severe sepsis or septic shock (see Chapter 86).
Cats
In cats, cat bite wounds may be difficult to identify on the sur-
face, but infection can extend to penetrate bone, joints, ten-
don and muscle.5 Cat bite wounds are most often located on
the forelimbs, lateral aspect of the face, and near the base of
the tail (Figure 57-3).8 In contrast, scratches are often found
on the bridge of the nose, pinna, and inguinal region. Cat bite
abscesses are characterized by the presence of firm or fluctu-
ant subcutaneous swellings or masses, with or without fever,
lethargy, inappetence, and hyperesthesia. Some cats are brought
to the veterinarian solely because of lethargy, and a careful
physical examination is required before an abscess or celluli-
tis is detected. One or more scabs may be found on top of the
abscess, or the overlying skin may lack hair and have a gray,
necrotic appearance. If the abscess ruptures, cream-colored or
red-brown purulent material may be identified on the haircoat,
sometimes in association with a foul odor (Figure 57-4). Alter-
natively, the haircoat may be matted over the site. Lameness
may be apparent if the limbs are involved, and especially if there
is extension to the bone or a joint. Involvement of the thoracic
cavity may be associated with fever, lethargy, tachypnea, and
decreased lung sounds due to pyothorax (see Chapter 87). Bite
wounds to the calvarium can result in neurologic signs such
as circling, disorientation, head pressing, mental obtundation,
delayed or absent placing reactions, tetraparesis, anisocoria,

FIGURE 57-3  Common anatomic distribution of cat-to-cat bite and fight wounds (“wound cat”). (Adapted from Malik R, Norris J, White J, et al. “Wound cat.” J Feline Med Surg
2006;8:135-140.)

A B
FIGURE 57-4  A, Six-year-old female spayed domestic shorthair cat with a cat bite abscess adjacent to the right eye that was associated with marked chemosis and periocular swelling.
B, Purulent material discharged from the abscess when it was drained surgically. (Images courtesy University of California, Davis Veterinary Ophthalmology service.)
550 SECTION 2  Bacterial Diseases

absent menace responses, and seizures.9 Penetration of the
caudal vertebral column and spinal cord can lead to vertebral
fractures, osteomyelitis, bacterial meningitis, and signs of pelvic
limb paralysis (Figure 57-5). As in dogs, progression to severe
sepsis or septic shock can occur, but most cat bite abscesses
rupture and resolve spontaneously.
Cats with dog bite wounds often have life-threatening inju-
ries. Fractures, severe hemorrhage, and hypovolemic shock
can dominate the clinical picture. Penetrating injury of cervical
structures, the thorax, abdominal organs, brain, or spinal cord
can occur (Figure 57-6). Death most often results from trauma
rather than infection, but occasionally deep-seated infections
or bacteremia develops if the cat survives the initial traumatic
episode.

FIGURE 57-5  Histopathology of the vertebral column of a young adult, male domes-
tic shorthair that was found with a bite wound to the base of the tail that drained purulent
material and pelvic limb paralysis with absent deep pain sensation. Multiple fractures
of caudal vertebrae 4 and 5 were present in association with suppurative osteomyeli-
tis (arrowheads), cellulitis, ascending meningitis, and masses of intralesional bacteria
(arrows). Multiple anaerobes were cultured from the lesion. H&E stain.
Diagnosis
A diagnosis of bite wound infections is based on history of a fight
or bite and physical examination findings that suggest that infec-
tion has developed. Cytologic examination of a wound may also
assist in diagnosis of infection. The veterinarian should record
whether the wound was provoked or unprovoked and note time
and location that the wound occurred and the biting animal
species involved. Depending on the extent and location of bite
wounds, radiographs of the affected region should be consid-
ered to assess for underlying damage to bone or body cavities.
All cats with cat fight wounds should be tested for FeLV and
FIV infection, and the test should be repeated 2 months later
because transmission may have occurred at the time of the fight
wound (see Chapters 21 and 22).
Laboratory Abnormalities
Laboratory abnormalities in cats or dogs with wound infec-
tions are variable and depend on the degree of tissue trauma,
the underlying organs involved (if any), and the severity and
type of infection. The CBC may show nonregenerative or regen-
erative anemia, neutrophilia with bandemia, toxic neutrophils,

B
FIGURE 57-6  A, Lateral radiograph of the lumbar spine of a 3-year-old female Siamese cat with a dog bite wound to the lumbar spine. The cat was unable to stand and had absent
placing reactions in the pelvic limbs. Patellar reflexes were also absent. Two osseous densities are superimposed at the level of the dorsal spinal canal at the level of the fourth lumbar ver-
tebra (L4) (arrow). The dorsoventral view showed deviation of the spinous process of L4 (not shown). CSF analysis revealed mild suppurative inflammation. B, Myelogram. A spinal needle
is inserted at L5-6 for injection of contrast agent. Spinal cord widening with associated thinning of the contrast column circumferentially is identified over the body of L4. Dorsal deviation
and splitting of the ventral contrast column is present over L2-3 as a result of disc protrusion in this region. A decompressive hemilaminectomy over L4 to L6 was performed and a bone
fragment was removed; the cat ultimately recovered.
 CHAPTER 57  Bite and Scratch Wound Infections
monocytosis, and lymphopenia or lymphocytosis. Animals with
severe sepsis or septic shock may be thrombocytopenic. Findings
on the chemistry panel include hypoalbuminemia and evidence
of muscle damage (increased activities of CK, AST, and some-
times mild increases in serum ALT activity). Increased muscle
enzyme activities may be a clue to an underlying cat bite wound
in cats with fever of unknown origin. Prolongations of the PT
and/or APTT may be present in animals with septic shock and
disseminated intravascular coagulation.
Diagnostic Imaging
Plain Radiography
Radiographs of the thorax in dogs or cats with bite wounds
may show fractured ribs, pneumothorax, evidence of pulmo-
nary contusions, mediastinal emphysema, diaphragmatic her-
niation, or pleural effusion due to hemothorax or pyothorax.
Abdominal radiographs may show a lack of serosal detail due
to hemoabdomen or bacterial peritonitis. Radiographs of the
axial or appendicular skeleton may reveal fractures or tooth for-
eign bodies. In more chronic bite wound infections, evidence of
osteomyelitis or septic arthritis may be present, with soft tissue
swelling and bony lysis (see also Chapter 85).
Sonographic Findings
Ultrasound of soft tissues can be useful to assess the extent of
bite wound infections. Abdominal ultrasound is indicated for
animals with bite wounds to the abdomen to assess for evidence
of peritonitis or trauma to abdominal organs. Findings in dogs
or cats with peritonitis include a hyperechoic mesentery with
an irregular outline and free peritoneal fluid, which is typically
echogenic (see Chapter 88).
Advanced Imaging
Findings on MRI in cats with brain abscesses secondary to
cat bite wounds include space-occupying lesions with well-
defined margins that are hyperintense on T2-weighted images,
and hypointense T1-weighted images.9 Marked ring enhance-
ment is usually present. Evidence of brain herniation may be
detected.9
Cytologic Examination
Cytologic examination of swab or fluid specimens collected
from infected wounds typically reveals large numbers of degen-
erate neutrophils with intracellular bacteria, which may include
cocci, rods, or long, filamentous organisms (which are usually
anaerobes).
Microbiologic Tests
Isolation and Identification: Dogs
Aerobic bacterial culture and susceptibility and anaerobic bac-
terial culture are indicated whenever possible from dogs that
are suspected to have infected bite wounds. Culture lacks sensi-
tivity for detection of anaerobes, and anaerobic bacterial infec-
tions respond predictably to treatment, so anaerobic culture
could be considered optional if client finances are limited (see
Chapter 37).
The skin around the wound should be clipped and cleaned
with chlorhexidine solution, and an aspirate, swab, or devital-
ized tissue specimen should be collected from as deep within the
wound as possible while the animal is sedated or anesthetized.
When pockets of purulent material extend subcutaneously away
from an open wound, collection of aspirates percutaneously
551
with a needle and syringe can avoid contamination by sur-
face bacteria. Because a positive culture from a wound does
not imply infection, results must be interpreted in light of the
physical examination and laboratory findings. Susceptibility test
results for isolated bacteria can then be used to guide antimi-
crobial drug treatment. Blood cultures should be considered for
dogs with evidence of severe sepsis or septic shock.
Isolation and Identification: Cats
In general, culture of cat bite abscesses in cats is not routinely
performed because they almost always respond to drainage with
or without systemic antibacterial drug treatment. Cytologic
examination and culture are indicated for cat bite abscesses/
cellulitis if clinical signs of infection are persistent or recur-
rent in the face of treatment, or if severe infections that involve
underlying bones, joints, or body cavities occur. Culture is also
recommended for draining skin lesions in the inguinal region,
which may result from infection with rapidly growing mycobac-
teria (see Chapter 44). Blood cultures are indicated for cats with
signs of severe sepsis or septic shock.
Treatment and Prognosis
Surgical Management: Dogs
Dogs with dog bite wounds should be muzzled and the wound(s)
covered with a clean, dry bandage immediately after the injury
occurs to prevent further contamination of the wound until
the animal’s condition can be stabilized if necessary. If possi-
ble, the dog should then be sedated or anesthetized and a wide
area of skin around the wound clipped in order to determine
the extent of injury and prepare for surgery. Frequently, this
leads to detection of additional full-thickness puncture wounds
or lacerations. The skin around the wound should be scrubbed
with chlorhexidine or povidone-iodine solution. Full-thickness
punctures and lacerations should then be explored, debris and
devitalized tissue removed, and specimens collected for culture
if infection is suspected. If the abdominal or thoracic cavity has
been penetrated, surgical exploration of these cavities is typically
required to identify and treat damaged viscera. Wounds should
be lavaged extensively with copious quantities of sterile saline
or lactated Ringer’s solution under moderate pressure (such as
using a 60-mL syringe attached to an 18-gauge needle). The use
of antibiotics or antiseptics to lavage the wound is not gener-
ally recommended, as they can be toxic to tissue and provide
minimal benefit for treatment of established wound infections.
If used, 0.05% chlorhexidine diacetate (1 part 2% chlorhexi-
dine to 39 parts water) is preferred because it has antibacterial
activity in the presence of organic debris, residual antibacterial
activity, and minimal systemic absorption and toxicity.
In general, infected wounds that are limited to the subcu-
taneous tissues and muscle are subsequently managed as open
wounds in order to optimize drainage and debridement. Closure
of infected wounds can lead to dehiscence and persistence of
infection. Whenever possible, the wound is covered with ban-
dages that absorb fluid, reduce dead space, prevent movement
and contamination of the wound, and assist in debridement.
Hydrophilic bandages are often used for the first 3 to 5 days.
Wound closure may be possible for noninfected wounds that
are in the “golden period” (first 6 to 8 hours after injury). The
reader is referred to small animal surgical texts for additional
information on surgical management of bite wound infections
and bandaging.10
552 SECTION 2  Bacterial Diseases
TABLE 57-2
Suitable Antimicrobial Drugs for the Treatment of Bite Wound Infections in Dogs and Cats*
Drug Species Dose (mg/kg)
Amoxicillin-clavulanic acid D, C 12.5-20
Ampicillin-sulbactam D, C 10-20†
Pradofloxacin‡ D 3
C 5-7.5
Enrofloxacin D 5-20
Marbofloxacin D 2.75-5.5
Gentamicin D 9-14
Amikacin D 15-30
Metronidazole D, C 10
C, Cat; D, dog.
*Doses of antimicrobial drugs that have activity primarily against gram-negative aerobes are listed for dogs, because isolation of gram-negative aerobes
from cat bite abscesses is very rare.
†Dose according to ampicillin component.
‡The pradofloxacin dose for dogs is for the tablet formulation available in Europe. The dose for cats is for the oral suspension. The use of pradofloxa-
cin in dogs may be associated with myelosuppression and is off-label in some countries.
Surgical Management: Cats
As for open dog bite wounds, a wide area of skin around cat bite
abscesses should be clipped and scrubbed. The abscess should
then be lanced and specimens collected for culture if desired.
The abscess cavity should then be lavaged, necrotic tissue
debrided, and a placement of a drain should be considered to
optimize drainage and prevent early wound closure. Whenever
possible, the wound and drain should then be covered with a
bandage. The drain is removed in 3 to 5 days. For cats with pyo-
thorax, placement of chest tubes with or without surgical explo-
ration may be required (see Chapter 87). Cats with abscesses
that involve the central nervous system have the potential to
recover with craniectomy, drainage of purulent material within
the abscess, removal of any tooth fragments present, and clo-
sure of subcutaneous tissue and the skin.9
Antimicrobial Treatment
Contaminated Wounds (First 8 Hours after Injury)
The need for antimicrobial treatment in contaminated, nonin-
fected wounds is controversial, because there is no evidence that
it prevents infection of the wound and it may select for infec-
tion with bacteria that are resistant to the antimicrobial drug
used. Proper wound cleaning and debridement are likely to have
the greatest impact on whether infection subsequently develops.
Antimicrobial treatment could be considered for deep punctures
or large, dirty wounds that require extensive debridement. If
deemed necessary, the drug of choice for contaminated wounds
is amoxicillin–clavulanic acid, because it has efficacy against
most oral cavity commensals of the dog and cat mouth such as
Pasteurella spp. and a variety of anaerobes, including those that
produce β-lactamase enzymes.
Infected Wounds
For severely infected dog bite wounds, initial treatment should
consist of parenteral antimicrobial drugs that have activity
against gram-negative and gram-positive aerobic and anaerobic
bacteria, which includes methicillin-resistant staphylococci (in
regions where methicillin-resistant S. pseudintermedius is prev-
alent). Appropriate combinations include ampicillin-­sulbactam
Interval (hours) Route
8-12 PO
8 IV, IM
24 PO
24 PO
24 IV, IM, PO
24 PO
24 IV, IM
24 IV, IM
8 PO, IV
and an aminoglycoside, or a combination of ampicillin, met-
ronidazole, and an aminoglycoside (Table 57-2). Where
methicillin-resistant S. pseudintermedius is not prevalent, a fluo-
roquinolone could be substituted for the aminoglycoside. Sub-
sequent treatment should be based on the results of culture and
susceptibility. Amoxicillin-clavulanic acid is the drug of choice
for treatment of cat bite abscesses once drainage has been estab-
lished (although many cat bite abscesses will likely heal with
drainage alone). Because Pasteurella spp. are not susceptible to
clindamycin or first-generation cephalosporins, monotherapy
with these drugs should be avoided. Pradofloxacin is also a suit-
able choice, because it has activity against anaerobes as well as
a variety of aerobic bacterial species.
Antimicrobial drug treatment of infected bite wounds that
are localized to the skin, subcutaneous tissue, and muscle
should continue for 3 to 5 days after signs of infection are no
longer evident. The reader is referred to Chapters 85, 87, and
88 for information on the management of osteomyelitis, septic
arthritis, pyothorax, and peritonitis.
Rabies Prophylaxis
In regions where rabies occurs, bite wounds may need to be
reported to local public health authorities. If the biting animal has
an unknown vaccination history, or the bite was from a wild ani-
mal that was unavailable for testing, a rabies booster should be
administered and the animal monitored for 45 days. If the bitten
animal is unvaccinated or has a lapsed vaccination status, addi-
tional quarantine or euthanasia may be required (see Chapter 13).
Prognosis
The prognosis for bite wound infections depends on the severity
and extent of injury, the immune status of the host, and the organ-
isms involved. Recurrent infections may result from underlying
osteomyelitis, foreign bodies, immunodeficiency, or the presence
of atypical organisms such as Mycobacterium spp., Nocardia
spp., or Mycoplasma spp. (which do not respond to β-lactam
drugs). Failure to respond to appropriate treatment should
prompt a thorough physical examination; testing for retroviruses
in cats; imaging of the affected area; and culture for aerobic and
 CHAPTER 57  Bite and Scratch Wound Infections
anaerobic bacteria and mycoplasmas. If the clinical picture is sug-
gestive, culture for mycobacteria may also be indicated.
Prevention
Prevention of bite wounds involves education of pet owners in
regard to housing cats indoors and use of secure collars and leashes
for walking dogs. Intact male dogs should be neutered if they are
not to be used for breeding. Proper handling and socialization of
puppies as well as behavior training should be encouraged. Pet
owners should be instructed to avoid or closely supervise inter-
actions between their dogs and unfamiliar dogs that are breeds
of known aggressive tendency, such as American pit bull terriers,
Rottweilers, German shepherds, blue heelers, chow chows, and
Jack Russell terriers. Special caution is warranted for small-breed
dogs, which may provoke larger breed dogs and are more likely
to develop life-threatening injuries. Dogs that are unfamiliar with
one another or dogs with a history of fighting with one another
should never be left together without proper supervision.
Public Health Aspects
Epidemiology of Dog and Cat Bite Wounds
In 2012, the American Pet Products Association estimated that
there are 78.2 million owned dogs and 86.4 million owned cats
in the United States, and that 39% of households own at least
one dog and 33% own at least one cat.11 In the United Kingdom,
around 22% of households own a dog and 18% own a cat.12 Ger-
man shepherds, Rottweilers, and Jack Russell terriers are among
the top 10 dog breeds owned. Dog and cat bite wounds account
for approximately 1% of emergency department visits by humans
in the United States, and similar numbers are reported in Europe.
Dog bites accounted for more than 70% and cat bites accounted
for 13% of animal bite visits to emergency rooms in New York
City.13 The median age of bitten people in one study was 15 years,
and in another study it was 28 years.14,15 Many bites occur in chil-
dren, especially boys aged 5 to 9 years.16 Children often have dog
bites to the face, neck, and head. Women and the elderly are at
risk for cat bite injuries. Patients who seek medical attention more
than 8 hours after injury usually have infected wounds.
Dog Bite Infections
Between 3% and 20% of dog bite wounds in humans become
infected.17,18 The infecting organisms generally reflect the flora
of the canine oral cavity, but may also derive from the environ-
ment or the bitten person’s skin or oral cavity. Many infections
are polymicrobial, but the organism that most often contributes
to dog bite wound infections is Pasteurella spp., which is found
in 50% of wounds (Table 57-3).14,19-27 The most common spe-
cies is P. canis, but P. multocida (subspecies multocida, septica,
and gallicida), P. stomatis, and P. dagmatis may also be present.
Other common isolates from dog bite wounds include strepto-
cocci (especially the α-streptococcus S. mitis and S. pyogenes;
S. canis is rarely isolated), staphylococci, Corynebacterium
spp., Neisseria spp., and anaerobes.19 Of the staphylococci,
S. aureus is isolated most often (20% of infections). Infections
with S. pseudintermedius occur rarely. Anaerobes are present in
approximately 70% of infections and are almost always present
in combination with other anaerobes and aerobic bacteria.
Cat Bite Infections
Although cat bite wounds are less traumatic to tissue, they
are more likely to become infected and progress to infection
553
more rapidly than dog bite wounds.14 The upper extremities
are often affected. As in dog bite wounds, Pasteurella spp. are
the most common organisms isolated and are present in 75% of
wounds.14,19 The predominant species are P. multocida subsp.
multocida and P. multocida subsp. septica. Streptococci (includ-
ing S. pyogenes) and staphylococci are also often present, but S.
aureus is isolated less frequently from cat bite wounds than dog
bite wounds. Neisseria and Moraxella are also often isolated
from cat bite wounds (see Table 57-3). The prevalence of infec-
tion with anaerobes in cat bite wounds is similar to that in dog
bite wounds.14
Clinical Manifestations
Infection of bite wounds in humans most often results in localized
cellulitis, pain, purulent discharge, a black to gray appearance,
and, less often, fever and local lymphadenopathy. Extension to
underlying bones or joints can lead to osteomyelitis or septic
arthritis, which is especially likely to occur after penetrating cat
bite wounds to the hand (Figure 57-7). Rarely, necrotizing fas-
ciitis, bacteremia, meningitis, endocarditis, peritonitis, or brain
abscesses develop.19,28
Most of the serious consequences of dog or cat bite wound
infections occur in immunocompromised humans and involve
P. multocida or Capnocytophaga canimorsus. Severe disease
manifestations have also occurred after exposure of wounds
to dog or cat saliva through licking, or after invasive medical
supplies (such as dialysis tubing) were chewed or licked by
a pet.29 In a few cases, patients have had a history of con-
tact with animals but no known bite or scratch wound.30-32
Life-threatening infections with P. multocida tend to occur
in infants, pregnant women with animal contact, patients
on chronic glucocorticoid therapy, HIV-infected people,
organ-transplant recipients, and patients with chronic liver
disease.28
Capnocytophaga canimorsus is a capnophilic (carbon
dioxide–loving), facultatively anaerobic, filamentous gram-
negative bacterial rod that is commonly found in the oral
cavity of dogs and cats.33 Although rarely reported as a
cause of illness in dogs or cats, this organism can cause cel-
lulitis, gangrene, bacteremia, meningitis, and endocarditis in
humans.28,33 C. canimorsus bacteremia is rare, but humans
who are immunocompromised, especially those with chronic
alcoholism, cirrhosis, or a history of splenectomy, are at risk
for life-threatening C. canimorsus infections that progress rap-
idly, with a mortality of 31%. The virulence factors that con-
tribute to these severe infections are not well understood. The
organism does not cause endotoxemia but appears to evade
recognition by the immune system and resist phagocytosis.28
In one series of humans with C. canimorsus sepsis, 56% of
cases followed a dog bite and 10% of cases followed expo-
sure of a wound to dog saliva by licking.34 Veterinarians have
also developed corneal infections with C. canimorsus when
dog tooth fragments have struck them in the eye during den-
tal extractions.35,36 Most affected humans are older than 40
years of age. Clinical manifestations of C. canimorsus sepsis
include fever, myalgia, and a petechial rash that involves the
skin and mucous membranes, which may progress from pur-
pura (purpura fulminans) to gangrene over a period of several
days. Because C. canimorsus is very difficult to grow, early
diagnosis and successful treatment require clinical suspicion
and early treatment with effective antimicrobial drugs, such as
β-lactam/β-lactamase inhibitor combinations, which are also
active against Pasteurella spp.
554 SECTION 2  Bacterial Diseases

TABLE 57-3
Pathogens That Can Infect Humans after a Bite from a Dog or Cat14,19-27
Dog Cat
Viruses Rabies Rabies
Aerobic Gram-positive bacteria Streptococcus (S. mitis)* Streptococcus (S. mitis)
Staphylococcus (S. aureus) Staphylococcus (S. epidermidis)
Corynebacterium (group G) Corynebacterium (C. aquaticum)
Enterococcus Enterococcus (E. durans)
Bacillus Bacillus (B. firmus)
Gemella morbillorum Actinomyces
Actinomyces viscosus Gemella morbillorum
Lactobacillus Erysipelothrix
Dermabacter hominis Rothia dentocariosa
Oerskovia Lactobacillus
Micrococcus lylae Rhodococcus spp.
Pediococcus Streptomyces spp.
Stomatococcus mucilaginosus
Aerobic Gram-negative bacteria Pasteurella (P. canis) Pasteurella (P. multocida)
Neisseria (N. weaveri) Neisseria (N. weaveri)
Moraxella Moraxella (M. catarrhalis)
Bergeyella zoohelcum Bergeyella zoohelcum
Capnocytophaga Capnocytophaga
Escherichia coli Acinetobacter
Brevibacterium Pseudomonas
Stenotrophomonas maltophilia Brevibacterium
Pseudomonas Gemella morbillorum
Klebsiella Actinobacillus
Citrobacter Alcaligenes
Flavobacterium Enterobacter
Eikenella corrodens Riemerella anatipestifer
Bartonella? Klebsiella oxytoca
Eikenella corrodens
Flavimonas oryzihabitans
Aeromonas hydrophila
Pantoea agglomerans
Bartonella
Francisella tularensis
Yersinia pestis
Anaerobic bacteria Fusobacterium (F. nucleatum) Fusobacterium (F. nucleatum)
Bacteroides (B. tectum) Bacteroides (B. tectum)
Prevotella (P. heparinolytica) Prevotella (P. heparinolytica)
Porphyromonas (P. macacae) Porphyromonas (P. gulae)
Propionibacterium (P. acnes) Propionibacterium (P. acnes)
Peptostreptococcus Peptostreptococcus
Tannerella forsythia Filifactor villosus
Eubacterium spp. Eubacterium spp.
Campylobacter spp. Clostridium sordellii
Veillonella spp.
Mycoplasmas Mycoplasma spp.
Mycobacteria Mycobacterium fortuitum Mycobacterium fortuitum
Mycobacterium kansasii
Fungi Blastomyces dermatitidis Sporothrix schenckii
Paecilomyces lilacinus

*For organisms that are present in >10% of cases, the most common species isolated is listed in parentheses.
 CHAPTER 57  Bite and Scratch Wound Infections
FIGURE 57-7  Cat bite wounds to the base of the thumb of a veterinarian with asso-
ciated cellulitis. Penetrating cat bite wounds of the hand have a tendency to extend to
underlying bones and joints.
CASE EXAMPLE
Signalment: “Murphy”, a 5-year-old intact male boxer dog
from Vacaville in northern California
History: Murphy was evaluated by the University of California,
Davis, veterinary emergency service for dog bite injuries after
a fight with another dog in the household. The owner had
returned home a few hours earlier to find Murphy bleeding
from the neck. The other dog, a 5-year-old male neutered
boxer mix, was found hiding and unable to stand with several
“scratches” on his body. Murphy had a history of fights with
other dogs. Both dogs in the household were fully vaccinated
for canine distemper virus, parvovirus, adenovirus, and rabies.
There was no other pertinent medical history.
Initial Physical Examination:
Body Weight: 46.2 kg.
General: Quiet, alert, responsive and hydrated. T = 102.1°F
(38.9°C), HR = 110, panting, pink and moist mucous
membranes, CRT = 1 s.
Integument: The haircoat was full, thick, and dirty. There was
a 1-cm superficial laceration on the ventral aspect of the
pinna, and approximately 10 puncture wounds on the left
ventral cervical region. A 2-cm superficial laceration was
present at the cranial aspect of the dorsal midline, and a
5-cm superficial laceration was present on the left flank. No
ectoparasites were seen.
Eyes, Ears, Nose, and Throat: Bilateral conjunctival hyperemia
and a mucoid ocular discharge were present. No other
significant abnormalities were noted.
555
Prevention
Veterinarians play an important role in educating the public
about prevention of cat and dog bite wounds. Provision of infor-
mation to clients who intend to purchase a new dog about dog
breeds, puppy socialization, dog behavior training, and how to
prevent and manage aggression and avoid bites or scratches (to
themselves and to others) is encouraged. Veterinarians can also
engage in community activities (especially in schools) where
young children can be educated about pet ownership and how
to avoid wounds and wound infections from their own or some-
one else’s dog or cat. Immunocompromised individuals should
be educated about the potential benefits and risks of pet owner-
ship and precautions that can be taken to minimize infections.
Finally, care should be taken in the veterinary practice situation
to avoid bites or scratches to veterinary staff. Veterinary staff
should be educated about the types of infections that can occur
in the workplace; likelihood of these infections; risk factors for
infection; appropriate preventative measures; and protocols to
follow should a bite or scratch wound occur (see General Ani-
mal Handling Precautions, Chapter 11). Routine hand-­washing
practices should be enforced, and licking by dogs or cats should
be discouraged. Face protection should be worn by veterinary
staff when dental procedures are performed. Bites and scratches
may need to be reported to public health authorities, and all
staff should be educated about the need to ensure that tetanus
and rabies immunizations are not overdue.
Musculoskeletal: BCS 6/9, ambulatory.
Cardiovascular, Respiratory, Gastrointestinal, Genitourinary,
Lymph Nodes, and Neurologic: No clinically significant
abnormalities were detected. Cranial nerve examination
was unremarkable. A full neurologic examination was not
performed.
Initial Treatment: Murphy was sedated with hydromorphone
and dexmedetomidine. The left neck was clipped with a no.
40 clipper blade from the midpoint of the dorsal aspect of
the head to the ventral aspect of the left ear and the tho-
rax. The skin was cleaned with 0.05% chlorhexidine solu-
tion, and all puncture sites were probed and flushed with
sterile saline. The sites were found to be relatively superfi-
cial. Drain sites were created with a no. 11 scalpel blade for
three wounds, and a 3 × 0.75 inch Penrose drain was placed
across a wound site that had the greatest amount of dead
space and secured with a single interrupted suture. The dog
was sent home with instructions for the owner to administer
amoxicillin-clavulanic acid (16 mg/kg PO q12h) and trama-
dol (2.4 mg/kg PO q8-12h), to monitor the wound for devel-
opment of excessive discharge, bleeding, swelling, or red-
ness, and to keep the area clean with a washcloth soaked in
warm water. Instructions were provided to return in 3 days
to have the wound reevaluated and the drain removed.
The owners returned 7 days later for drain removal. At that time,
they reported that Murphy had been eating normally, but
had been drinking more than usual. His activity level had
been normal, but they believed his neck was had become
swollen and tender. They had been administering the medi-
cations as directed.
Continued
556 SECTION 2  Bacterial Diseases
Physical Examination (Day 7):
Body Weight: 46.2 kg.
General: Quiet, alert, responsive, and hydrated but anxious. T =
103.1°F (39.5°C), HR = 114, RR = 36, pink and moist mucous
membranes, CRT 1 s.
Integument: There were marked crusts and scabs on the left
lateral and ventral aspect of the neck. The Penrose drain
was present with minimal amounts of discharge. Serous to
purulent discharge was draining from the ventral puncture
wounds. There was moderate crusting of the dorsal aspect
of the left pinna.
Other Systems: No change from the previous examination.
Treatment (Day 7): The dog was again sedated and purulent
material was collected for culture and susceptibility. The
eschars were gently removed with forceps and the skin
scrubbed with 0.05% chlorhexidine and water. Ultrasound
of the neck region revealed pockets of hypoechoic material
beneath the skin, and surgical debridement of the area
under general anesthesia was recommended. Subsequently
Murphy was anesthetized and the wound was explored. No
abscesses were found. The devitalized tissue was excised,
the wounds flushed vigorously, and two Penrose drains were
placed. The neck was bandaged initially, but this interfered
with the dog’s ability to breathe comfortably, so the
bandage was removed. The dog was hospitalized overnight
and treated with hydromorphone (0.05 mg/kg, IV, q4h) and
warm compresses to the neck (q6h for 5 minutes). He was
then sent home with instructions to administer tramadol as
previously prescribed, clindamycin (20 mg/kg PO q12h), and
enrofloxacin (10 mg/kg PO q24h), and to warm compress
the neck every 4 to 6 hours for the next 3 days. They were
also instructed to return in 3 to 5 days for reevaluation and
drain removal.
Microbiologic Testing:
Direct Smear and Gram Stain: Rare numbers of gram-positive
cocci.
Aerobic and Anaerobic Culture and Susceptibility (Swab
from Wound): Isolate 1: Methicillin-resistant Staphylococcus
aureus, resistant to all β-lactams and clindamycin;
susceptible to amikacin (≤4 µg/mL), chloramphenicol (8
µg/mL), doxycycline (≤2 µg/mL), enrofloxacin (≤0.25 µg/
mL), gentamicin (≤1 µg/mL), marbofloxacin (≤0.25 µg/mL),
rifampin (≤1 µg/mL), and trimethoprim-sulfamethoxazole
(≤0.50 µg/mL).
Isolate 2: Enterococcus faecium (intrinsic resistance to low-
level aminoglycosides, cephalosporins, clindamycin, fluo-
roquinolones, trimethoprim-sulfamethoxazole), resistant
to chloramphenicol and rifampin. Intermediate suscepti-
bility to doxycycline (8 µg/mL), erythromycin (1 µg/mL),
ticarcillin (64 µg/mL), and ticarcillin-clavulanate (64 µg/
mL). Susceptible to ampicillin (1 µg/mL), clavulanic acid–
amoxicillin (≤4 µg/mL), penicillin (4 µg/mL), and imipenem
(≤1 µg/mL).
Isolate 3: Pseudomonas aeruginosa, resistant to ampicillin,
amoxicillin-clavulanic acid, all cephalosporins tested, chlor-
amphenicol, and doxycycline. Susceptibility to clindamycin
was not reported because this species is usually resistant.
Susceptible only to amikacin (≤4 µg/mL), gentamicin (2 µg/
mL), enrofloxacin (0.50 µg/mL), imipenem (≤1 µg/mL), ticar-
cillin (16 µg/mL), ticarcillin–clavulanic acid (16 µg/mL). The
MICs for trimethoprim-sulfamethoxazole and marbofloxacin
were 2 µg/mL and ≤0.25 µg/mL, respectively.
Anaerobic culture: Moderate numbers of mixed growth that
included Bacteroides ureolyticus group (β-lactamase nega-
tive), Peptostreptococcus anaerobius, and Bacteroides
spp./Prevotella spp. (β-lactamase negative).
Diagnosis: Polymicrobial bite wound infection with anaerobes
and multiple drug-resistant aerobic gram-positive and
gram-negative bacteria.
Outcome: On receipt of the culture and susceptibility test
results, the clindamycin was substituted with amoxicillin-
clavulanic acid (16 mg/kg PO q12h). Treatment with
enrofloxacin was continued and the owners were
educated in regard to the public health significance of the
infection and the need to wear gloves when handling the
wound and for scrupulous hand-washing practices. The dog
returned 4 days after surgery for drain removal. The owners
reported that the drains had been producing considerable
amounts of purulent material and that Murphy was doing
well. He was afebrile and the wound was unchanged in
appearance. He was again sedated, the area was scrubbed,
and crusts were removed. The drains were removed. Murphy
recovered uneventfully and the sutures were removed 10
days later at a local veterinary clinic. Five months later he
was seen again for multiple bite wounds to the thoracic
limbs and right thorax.
Comments: In this dog, bite wound injuries were complicated
by infection with multiple gram-negative and gram-positive
drug-resistant aerobic bacteria as well as anaerobes. The
S. aureus and P. aeruginosa were resistant to amoxicillin-
clavulanic acid, which had been used to prevent infection
when the dog was first seen. In contrast, the anaerobes and
the E. faecium were susceptible to amoxicillin-clavulanic
acid, but presumably were able to proliferate and persist
in the face of antimicrobial drug treatment as a result of
the presence of the other drug-resistant bacteria, purulent
material, and dead space. Bite wounds to the neck can be
a challenge to manage because of the mobility of this area,
which contributes to the maintenance of the dead space.
The use of a bandage can reduce dead space, avoid further
contamination, and reduce exposure to multidrug-resistant
bacteria, but attempts to bandage the wound were not
tolerated by this dog. Client education was an important part
of management. The owners were instructed to separate the
dogs when unattended to avoid further fights. Neutering
may also have been of benefit.
 CHAPTER 57  Bite and Scratch Wound Infections
SUGGESTED READINGS
Gaastra W, Lipman LJ. Capnocytophaga canimorsus. Vet Microbiol.
2010;140:339-346.
Love DN, Malik R, Norris JM. Bacteriological warfare amongst cats:
what have we learned about cat bite infections? Vet Microbiol.
2000;74:179-193.
Malik R, Norris J, White J, et  al. Wound cat. J Feline Med Surg.
2006;8:135-140.
Oehler RL, Velez AP, Mizrachi M, et  al. Bite-related and septic syn-
dromes caused by cats and dogs. Lancet Infect Dis. 2009;9:439-447.
REFERENCES
1. Griffin GM, Holt DE. Dog-bite wounds: bacteriology and treatment
outcome in 37 cases. J Am Anim Hosp Assoc. 2001;37:453-460.
2. Meyers B, Schoeman JP, Goddard A, et al. The bacteriology and
antimicrobial susceptibility of infected and non-infected dog bite
wounds: fifty cases. Vet Microbiol. 2008;127:360-368.
3. Mouro S, Vilela CL, Niza MM. Clinical and bacteriological assess-
ment of dog-to-dog bite wounds. Vet Microbiol. 2010;144:127-132.
4. Love DN, Jones RF, Bailey M, et al. Isolation and characterisation
of bacteria from abscesses in the subcutis of cats. J Med Microbiol.
1979;12:207-212.
5. Love DN, Malik R, Norris JM. Bacteriological warfare amongst
cats: what have we learned about cat bite infections? Vet Micro-
biol. 2000;74:179-193.
6. Love DN, Jones RF, Bailey M, et al. Bacteria isolated from subcu-
taneous abscesses in cats. Aust Vet Practit. 1978;8:87-90.
7. Norris JM, Love DN. The isolation and enumeration of three feline
oral Porphyromonas species from subcutaneous abscesses in cats.
Vet Microbiol. 1999;65:115-122.
8. Malik R, Norris J, White J, et al. Wound cat. J Feline Med Surg.
2006;8:135-140.
9. Costanzo C, Garosi LS, Glass EN, et al. Brain abscess in seven cats
due to a bite wound: MRI findings, surgical management and out-
come. J Feline Med Surg. 2011;13:672-680.
10. Fossum TW, Hedlund CS, Johnson AL, et al. Surgery of the integu-
mentary system. In: Fossum TW, ed. Small Animal Surgery. 3rd ed.
St. Louis, MO: Mosby; 2007:159-259.
11. The Humane Society of the United States. 2012. http://www.
humanesociety.org/issues/pet_overpopulation/facts/pet_ownership_
statistics.html. Last accessed January 1, 2013.
12. Pet Population 2011. 2011. http://www.pfma.org.uk. Last accessed
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duct dog and animal bite surveillance in New York City, 2003-
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14. Talan DA, Citron DM, Abrahamian FM, et al. Bacteriologic analy-
sis of infected dog and cat bites. Emergency Medicine Animal Bite
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15. Weiss HB, Friedman DI, Coben JH. Incidence of dog bite injuries
treated in emergency departments. JAMA. 1998;279:51-53.
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Rep. 2003;52:605-610.
17. Goldstein EJ. Bite wounds and infection. Clin Infect Dis.
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18. Underman AE. Bite wounds inflicted by dogs and cats. Vet Clin
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19. Abrahamian FM, Goldstein EJ. Microbiology of animal bite wound
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20. Zendri E, Martignoni G, Benecchi M, et al. Paecilomyces lilacinus
cutaneous infection associated with a dog bite. J Am Acad Derma-
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21. Ngan N, Morris A, de Chalain T. Mycobacterium fortuitum infec-
tion caused by a cat bite. N Z Med J. 2005;118:U1354.
22. Southern Jr PM. Tenosynovitis caused by Mycobacterium kansasii
associated with a dog bite. Am J Med Sci. 2004;327:258-261.
23. Ariel I, Haas H, Weinberg H, et  al. Mycobacterium fortuitum
granulomatous synovitis caused by a dog bite. J Hand Surg Am.
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24. Gnann Jr JW, Bressler GS, Bodet 3rd CA, et al. Human blastomy-
cosis after a dog bite. Ann Intern Med. 1983;98:48-49.
25. Thornton DJ, Tustin RC, Pienaar BJ, et  al. Cat bite transmis-
sion of Yersinia pestis infection to man. J S Afr Vet Assoc.
1975;46:165-169.
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27. McCabe SJ, Murray JF, Ruhnke HL, et al. Mycoplasma infection of
the hand acquired from a cat. J Hand Surg Am. 1987;12:1085-1088.
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dromes caused by cats and dogs. Lancet Infect Dis. 2009;9:439-447.
29. Boinett C, Gonzalez A. Pasteurella multocida septicaemia in a
patient on haemodialysis. BMJ Case Rep. 2009.
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two cats. Transfusion. 2007;47:1984-1989.
31. Waghorn DJ, Robson M. Occupational risk of Pasteurella multo-
cida septicaemia and premature labour in a pregnant vet. BJOG.
2003;110:780-781.
32. Wade T, Booy R, Teare EL, et  al. Pasteurella multocida men-
ingitis in infancy-(a lick may be as bad as a bite). Eur J Pediatr.
1999;158:875-878.
33. Gaastra W, Lipman LJ. Capnocytophaga canimorsus. Vet Micro-
biol. 2010;140:339-346.
34. Pers C, Gahrn-Hansen B, Frederiksen W. Capnocytophaga cani-
morsus septicemia in Denmark, 1982-1995: review of 39 cases.
Clin Infect Dis. 1996;23:71-75.
35. de Smet MD, Chan CC, Nussenblatt RB, et  al. Capnocytophaga
canimorsus as the cause of a chronic corneal infection. Am J
­Ophthalmol. 1990;109:240-242.
36. Chodosh J. Cat’s tooth keratitis: human corneal infection with
Capnocytophaga canimorsus. Cornea. 2001;20:661-663.
 SECTION 3
Fungal and Algal Diseases
CHAPTER 58
Dermatophytosis
Jane E. Sykes and Catherine A. Outerbridge
Overview of Dermatophytosis
First Described: Dermatophytosis was described indepen-
dently by several scientists in the 1830s and 1840s,1 but
probable descriptions of ringworm date back to ancient
Greek times. Dermatophytes were more fully character-
ized in the early 1900s by Sabouraud.2
Causes: Microsporum and Trichophyton species (especially
Microsporum canis, Microsporum gypseum, and Trichophy-
ton mentagrophytes) (teleomorph name, Arthroderma spp.)
Geographic Distribution: Worldwide, especially warm and
humid climates
Mode of Transmission: Direct contact with other infected
hosts, fomite transmission
Major Clinical Signs: Cutaneous lesions include alopecia, ery-
thema, crusting and scaling, and sometimes nodular or
draining lesions (kerions and dermatophytic mycetomas).
Differential Diagnoses: Alopecic lesions must be differentiated
from self-traumatic alopecia secondary to allergic dermati-
tis, sarcoptic mange, or demodicosis. If scale and crusting
are also present, bacterial folliculitis, superficial pyoderma,
and pemphigus foliaceus are differential diagnoses. Nodu-
lar lesions must be differentiated from eosinophilic granu-
loma, feline acne (cats), and mast cell tumors (dogs).
Human Health Significance: Dermatophytosis is a zoono-
sis. Young children and the immunocompromised can
develop extensive cutaneous lesions.
Etiology and Epidemiology
Dermatophytosis (ringworm or tinea) is a superficial cutaneous
infection with one or more of the keratinophilic fungi that belong
to the genera Microsporum, Trichophyton, or E ­ pidermophyton.
Transmission of dermatophytes occurs by close contact with
558
other infected animals or through contact with contaminated
fomites (which includes the haircoats of animals and arthro-
pods such as fleas or houseflies). Dermatophyte spores survive
more than a year in the environment under optimal conditions
of temperature and humidity, and they resist most routinely
used hospital disinfectants, which facilitates transmission. Der-
matophytes are somewhat host species specific and are classified
as geophilic, zoophilic, or a­ nthropophilic (Table 58-1).3-8 Geo-
philic dermatophytes are soil saprophytes. The most common
geophilic dermatophyte that infects dogs or cats is ­Microsporum
gypseum, which is most prevalent in warm, humid tropical and
subtropical environments. Zoophilic dermatophytes are adapted
to animal hosts and are rarely found in soil. The most common
zoophilic dermatophyte that infects dogs and especially cats is
Microsporum canis; this organism accounts for more than 90%
of dermatophyte isolates from cats worldwide and more than
60% of isolates from dogs.3,9,10 Sylvatic dermatophytes are zoo-
philic dermatophytes that are adapted to rodents or hedgehogs.
The most common sylvatic dermatophyte that infects dogs and
cats is Trichophyton ­mentagrophytes. Anthropophilic derma-
tophytes are adapted to human hosts and do not survive in the
soil; they include ­Microsporum ­audouinii, Trichophyton ton-
surans, ­Trichophyton rubrum, and Epidermophyton ­floccosum.
Rarely, these species infect or contaminate dogs or cats that
have a history of close contact with infected humans.4,11-16
Mixed infections with multiple dermatophyte species occur
rarely in dogs.10
Risk factors for dermatophytosis in dogs and cats include
young age and concurrent immunosuppressive disorders,
especially endogenous or iatrogenic hyperadrenocorticism.
Dermatophytosis is more common in cats than in dogs, but
the prevalence varies with geographic location. Positive der-
matophyte cultures occurred in 6% of submissions from dogs
or cats in the southern United States (3.8% for dogs and
14.9% for cats),17 and in the United Kingdom, 16% were pos-
itive (10% for dogs and 16% for cats).9 Higher prevalences of
detection (19% to 43%) have been reported in some studies
 CHAPTER 58  Dermatophytosis 559

TABLE 58-1
Zoophilic and Geophilic Dermatophyte Species Reported to Infect Dogs and/or Cats3-8
Dermatophyte Species Classification Primary Reservoir Species Affected Geographic Distribution
Microsporum canis Zoophilic Cats, dogs, horses All mammals Temperate and tropical
locations worldwide
Microsporum gypseum Geophilic Soil All mammals Worldwide
Microsporum persicolor* Zoophilic Voles, field mice Dogs, cats Europe
Trichophyton mentagrophytes Zoophilic Rodents All mammals Worldwide
Trichophyton erinacei Zoophilic Hedgehogs Dogs Europe and New Zealand
Trichophyton simii Zoophilic Primates Dogs, cats India and the Far East
Trichophyton terrestre* Geophilic Soil All mammals Worldwide

*Do not invade hair; the role of T. terrestre in disease causation is controversial.

of cats and dogs, with and without skin lesions, from parts
of continental Europe.3,5,10 Dermatophytosis (especially geo-
philic dermatophytosis) is more prevalent in regions with high
warmth and humidity. Shelter cats, the vast majority of which
lack skin lesions, are more likely to carry dermatophytes on
their haircoats than healthy pet cats. Dermatophytes were
cultured from 5.5% and 19% of all shelter cats depend-
ing on geographical region,18,19 whereas the prevalence of
M. canis isolation from healthy pet cats is generally less than
2.5%.18 Animals admitted to breeding or boarding facili-
ties that have a history of dermatophytosis are also at risk
of infection, whereas dermatophytes are generally not detect-
able on the haircoats of animals in facilities without a his-
tory of infection.20 Retrovirus-infected cats are no more likely
to carry dermatophytes than retrovirus-negative cats,21 but
retrovirus-infected cats carry a greater range of species and
may be more likely to develop skin lesions (dermatophytosis).
Genetic factors may also be important. Persian and Himala-
yan cats and Yorkshire and Jack Russell terriers appear to
be predisposed to dermatophytosis.3,9,17 In particular, Persian
cats are thought to be at greater risk for development of der-
matophytic mycetomas (see Clinical Features). Dogs that hunt
A
and burrow in soil may be at increased risk for infection by
geophilic or sylvatic dermatophytes, which may explain the
common distribution of lesions on the face and distal thoracic
limbs for these dermatophyte species (Figure 58-1).

Clinical Features
Signs and Their Pathogenesis
Pathogenic dermatophytes invade keratinized tissue that has
been macerated or traumatized. Infections are localized to the
stratum corneum, hair shafts, and claws. The clinical expres-
sion of the disease is affected by the fungal species and strain
involved and the host response to infection.22 Infectious arthro-
spores, which are formed by the segmentation and fragmenta-
tion of fungal hyphae, adhere to the surfaces of skin and nails,
germinate, and secrete proteases. These proteases, such as sub-
tilisins, fungalysins, and a variety of acidic proteases,22,23 digest
keratin into short peptides or amino acids, which are then used B
by the fungus as a source of nutrition. The pattern of prote- FIGURE 58-1  Chronic Trichophyton mentagrophytes infections. There is alope-
ases secreted by different dermatophyte strains may influence cia, crusting, hyperpigmentation, and ulceration of the muzzle and periocular skin. 
the clinical expression of disease. On the host, dermatophytes A, Four-and-a-half-year-old male neutered fox terrier. B, Six-year-old female spayed Jack
reproduce asexually through production of conidia. Russell terrier. (Courtesy University of California, Davis, Veterinary Dermatology Service.)
560 SECTION 3  Fungal and Algal Diseases
A B
FIGURE 58-2  A, Jack Russell terrier puppy with multifocal classic well-circumscribed lesions caused by infection by Microsporum canis. B, On questioning, the owner of the affected
litter of puppies revealed that she also had a lesion on her arm. (Courtesy Dr. Terry Nagle.)
FIGURE 58-3  Nine-year-old female spayed labrador retriever with multiple kerions
on the muzzle due to Microsporum canis infection. Another dog in the household had
similar lesions that were less severe. (Courtesy University of California, Davis, Veterinary
Dermatology Service.)
Dermatophyte lesions form after an incubation period of 1
to 3 weeks. Classically, lesions are single or multiple and consist
of circular regions of alopecia with an erythematous margin,
superficial scale and crust, and follicular papules and pustules
(Figure 58-2). Central healing may occur, sometimes with
hyperpigmentation or hair regrowth in the center of the lesion.
Pruritis is variable. Infection by T. mentagrophytes in dogs
can result in progressive alopecia, with dramatic scaling and/
or crusting and inflammation and in some cases scarring (see
Figure 58-1). A kerion or kerion reaction (also known as nodu-
lar dermatophytosis) is a localized, nodular inflammatory lesion
that results from dermatophyte infection and may be associated
with infection by M. gypseum, T. mentagrophytes, or M. canis
(Figure 58-3).10,24,25
Longhaired cats may develop a poor haircoat, shed exces-
sively, and vomit hairballs more frequently, because infected
hair shafts are friable and prone to breakage. Persian and
Himalayan cats (and very rarely, domestic longhair cats and
dogs) with M. canis infections can develop dermatophytic
mycetomas, which are coalescing subcutaneous nodular
lesions that often drain purulent fluid (Figure 58-4).26-31 These
develop when dermatophytes invade the dermis and subcuta-
neous tissues, presumably after traumatic implantation of fun-
gal organisms from the haircoat of an already colonized or
infected animal. These lesions have also been referred to as
granulomatous dermatitis, Majocchi’s granuloma, or “pseu-
domycetomas,” although use of the last term was not recom-
mended by the International Society for Human and Animal
Mycology (ISHAM) committee for nomenclature of fungal
diseases.32 In contrast to true mycetomas (eumycetomas), der-
matophytic mycetomas are not caused by soil saprophytes, and
lesions are often multiple and coalescing, rather than single
and localized.10
Physical Examination Findings
In dogs, dermatophytosis can occur anywhere on the body but
most often occurs on the face, distal limbs, and tail. Dogs are
more likely than cats to present with classical, localized, well-
circumscribed lesions. Claw infection is manifested by erythema
and thickening of the ungual fold, deformity and friability of the
claw, and sometimes footpad involvement (Figure 58-5). Kerions
most commonly develop on the muzzle or distal limbs, and with
digital pressure they typically have a boggy or exudative appear-
ance (see Figure 58-4).
Lesions in cats are extremely pleomorphic and can consist
of one or more areas of partial alopecia with scale and crusts,
especially on the pinnae and pinnal margins, nasal bridge, distal
limbs, or tail (Figure 58-6). Some lesions resemble miliary der-
matitis or are focal, pruritic lesions that resemble eosinophilic
plaques. Cats with dermatophytic mycetomas have focal (and
rarely disseminated) nodular, sometimes ulcerated cutaneous
lesions, usually on the dorsum or neck, which may drain serous
or purulent fluid (see Figure 58-4).

Diagnosis
Diagnosis of dermatophytosis is based on clinical suspicion
together with the results of Wood’s lamp examination, skin and
hair cytology, skin scrapings (to evaluate for demodicosis), and/or
fungal culture (Table 58-2). In both dogs and cats, lesions are rarely
symmetric. This asymmetry can help differentiate dermatophyto-
sis from other diagnoses that more commonly cause symmetrical
lesions. However, diagnosis that is solely based on the appearance
of lesions and/or Wood’s lamp examination is not recommended,
because it can lead to misdiagnosis and unnecessary treatment that
may be expensive and even harmful. In addition, mixed processes
can occur, such as co-infections with dermatophytes and staphy-
lococci or dermatophytes and Demodex mites. Because dermato-
phytosis can take on a wide range of appearances in cats, it should
be considered a possible differential diagnosis in cats with any skin
disease.33
Laboratory Abnormalities
Complete Blood Count, Serum Biochemical Tests, and Urinalysis
The results of routine laboratory testing in dogs and cats
with dermatophytosis are typically unremarkable or show
B
FIGURE 58-4  A and B, Twelve-year-old female spayed Himalayan with a derma-
tophytic mycetoma. Ulceration and fistulation are present. Ultimately, surgical excision
of the mycetoma was required. (Courtesy University of California, Davis, Veterinary
Dermatology Service.)
CHAPTER 58  Dermatophytosis 561
abnormalities associated with predisposing disease condi-
tions (such as hyperadrenocorticism).
Microbiologic Tests
Wood’s Lamp Examination
A positive Wood’s lamp examination is characterized by the pres-
ence of apple-green fluorescence along the length of a hair shaft
(Figure 58-7). The fluorescence results from tryptophan metabo-
lites produced by some M. canis strains when they grow within the
hair shaft; the metabolite is not produced when the organism grows
on scale or the claw. A significant proportion of M. canis infections
do not fluoresce,3,9 and other dermatophyte species of veterinary
significance do not produce fluorescent metabolites, so the sensi-
tivity of Wood’s lamp examination is low. The lamp should be
allowed to warm up for several minutes before it is used, and the
lights should be turned off during the examination. False-positive
results can occur if topical ointments, scale, lint, keratinized debris,
or sebum are present and incorrectly interpreted as dermatophyte
fluorescence. Hairs that fluoresce can be plucked and submitted for
cytologic examination and fungal culture.
Cytologic Examination
Cytologic examination of hairs plucked from lesions or the cir-
cumference of lesions may reveal fungal hyphae or chains of
arthroconidia (ectothrix spores) in some affected animals; mac-
roconidia, which form in culture, are not seen (Figure 58-8).
Addition of a clearing agent such as 10% or 20% potassium
hydroxide (KOH) or chlorphenolac is recommended, which
removes scale and debris that can interfere with organism iden-
tification. Potassium hydroxide solutions must be gently heated
with the hair for 10 minutes for clearing to occur, or incubated
FIGURE 58-5  Foot of a 9-year-old intact male terrier mix with dermatophyto-
sis caused by Microsporum gypseum. The dog competed in rodent hunting events that
occurred in underground tunnels. Severe, chronic dermatitis with ulceration and alopecia
that involved multiple distal limbs and claws was present. The claws were irregular and
discolored. (Courtesy University of California, Davis, Veterinary Dermatology Service.)
562 SECTION 3  Fungal and Algal Diseases

overnight at room temperature. Chlorphenolac, which is com-

posed of chloral hydrate, phenol, and lactic acid, is not readily
available and is toxic, but it does not require heating or pro-
longed incubation. Examination of hairs mounted in mineral oil
can still yield positive results in the absence of a clearing agent.25
Cytologic examination has low sensitivity when compared
with culture, and experience is required for proper organism
identification.

A
B color. The DTM turns red when the first dermatophyte colony
FIGURE 58-6  Eighteen-year-old male neutered domestic shorthair cat with dermato-
phytosis caused by Trichophyton mentagrophytes. The paws (A), pinnae (B), and nasal bridge
were involved. (Courtesy University of California, Davis, Veterinary Dermatology Service.)
Fungal Culture
Culture is the gold standard for diagnosis of dermatophytosis.
Ten to 12 hairs (ideally those that appear damaged or broken)
should be plucked from the edge of lesions with sterile forceps
and placed in a clean, dry paper envelope (preferably) or a ster-
ile tube or container at room temperature. Scrapings of crusts
from the edge of lesions or nail clippings (after cleaning the nail
with 70% alcohol) can also be submitted (see also Chapter 4 for
specimen collection methods for dermatophytes). Alternatively,
a brand-new toothbrush (out of the package) can be used to
collect specimens from lesions or the entire coat of a suspect ani-
mal by vigorous brushing for several minutes. The toothbrush
is then submitted to the laboratory. Macerated tissue cultures
can be performed on biopsy specimens from animals that have
nodular dermatophytosis or dermatophytic mycetomas.
The most commonly used media for culture are Dermato-
phyte Test Medium (DTM) or Sabouraud’s dextrose agar. In-
house DTM cultures, if performed, should be interpreted with
caution. Flat Petri plates (as opposed to vials) should be used in
order to facilitate inoculation and species identification. Inocu-
lated media should be incubated at 24°C to 30°C; incubation at
room temperature is more likely to result in false negatives.34
Growth occurs within 14 days, and often within 5 to 7 days,
but may take up to 21 days (particularly for treated animals).
Dermatophyte colonies, which are white or cream and powdery
(Figure 58-9), should not be confused with those of saprophytic
molds, which may be brown, green, black, gray, or mixed in
is visible. Color changes that occur after the first colony appears
may result from growth of saprophytic fungi. All suspicious col-
onies must be confirmed as dermatophytes and speciated based

TABLE 58-2
Diagnostic Assays Available for Dermatophytosis
Assay Specimen Type Target Performance
Wood’s lamp Haircoat Fluorescent Low sensitivity (around 50%) for detection of M. canis when
examination Microsporum performed properly. Other dermatophyte species do not fluo-
canis strains in resce. The presence of scale, sebum, debris, and topical oint-
hair ments may lead to false positive results. When positive, allows
identification of suitable specimens for cytology and/or culture.
Cytology Hair pluck specimens Dermatophyte When positive, allows early institution of treatment. Low
hyphae and sensitivity. Incubation with clearing agents may increase
arthrospores sensitivity, but these may not be readily available. Other
fungal conidia that contaminate the haircoat or debris may
be mistaken for dermatophyte elements.
Culture Plucked hair specimens, All dermatophyte Gold standard for diagnosis of dermatophytosis. Prolonged
scale and nail clippings species incubation times may be required.
Histopathology Skin biopsies Dermatophytes Organisms may be visualized with silver stains, periodic
acid–Schiff stain, or immunohistochemistry.
 CHAPTER 58  Dermatophytosis 563

on spore morphology through the use of microscopy (see Figure

58-9). Spores form after 7 to 10 days of growth. The individual
performing the identification should wear gloves. Tape is placed
over the colony and transferred to a slide, on which a drop of
stain is applied. A coverslip is then applied. Lactophenol cotton
blue or new methylene blue are suitable stains. Dysgonic strains
of M. canis have been described that have atypical morphology
in culture, but these are rare.27,35 Whenever possible, submis-
sion to a veterinary diagnostic laboratory that follows Clinical
and Laboratory Standards Institute (CLSI) guidelines is rec-
ommended to ensure proper quality assurance for culture and
identification of dermatophytes. This also minimizes the risk of
laboratory-acquired infections and contamination of the prac-
tice environment. Matrix-assisted laser desorption/ionization–
time of flight mass spectrometry (MALDI-TOF) offers promise
for rapid identification of dermatophytes in veterinary diagnos-
tic microbiology laboratories in the future.36

FIGURE 58-7  Positive Wood’s lamp examination in a 2-year-old male neutered
Persian that was seen for pruritis and hair loss. (Courtesy University of California, Davis,
Veterinary Dermatology Service.)
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays have been developed that detect and differentiate
between dermatophytes that infect dogs and cats.37,38 Sensitive
real-time PCR assays have been described for use in humans,39,40
but their use has not been reported for the diagnosis of derma-
tophytosis in dogs or cats. PCR assays have the potential to be

A B

C
FIGURE 58-8  Cytology images of hairs infected with dermatophytes. A, Unstained preparation. Large numbers of Microsporum canis spores are seen that surround the hair shaft.
B, Parker ink-KOH stain. Arthrospores and hyphae can be seen. C, Unstained preparation of a hair infected by Microsporum gypseum.
564 SECTION 3  Fungal and Algal Diseases
more sensitive than culture, with a more rapid turnaround time,
and have the potential to facilitate assessment of treatment effi-
cacy and control of disease in group-housed animals.
Pathologic Findings
Histopathology of biopsy specimens from dogs or cats with
dermatophytosis may reveal spores and hyphae within intrafol-
licular hair shafts, in association with a variable pyogranuloma-
tous inflammatory response (Figure 58-10). Special stains such
as periodic acid–Schiff (PAS) or Gomori’s methenamine silver
can increase the sensitivity for detection of fungal elements.41
Immunohistochemical stains have also been used to detect
M. canis within lesions.26 Occasionally, dermatophytes produce
a superficial pustular histologic lesion. Acantholytic keratino-
cytes can be seen in biopsies from some dogs, which may lead
to misdiagnosis of pemphigus foliaceus.41 Histopathology of
representative skin biopsies is especially useful for diagnosis
of dermatophytic mycetomas, kerions, and fungal paronychia,
because organisms are typically present in low numbers in these
lesions and culture may be negative. Histopathology of derma-
tophytic mycetomas reveals pyogranulomatous inflammation
B
FIGURE 58-9  Selected cultural characteristics of dermatophytes. A, Microsporum
canis from a cat with a dermatophytic mycetoma growing on inhibitory mold agar.
B, Macroconidia of M. gypseum after growth in culture.
and PAS-positive fungal elements that are embedded in an
eosinophilic amorphous material within the dermis.
Treatment and Prognosis
Most otherwise healthy animals with dermatophytosis clear the
infection without specific antifungal treatment within 3 months of
diagnosis. Nevertheless, treatment is strongly recommended for
animals that reside in multianimal households and those that live
with immunocompromised humans (which includes children and
older people, e.g., 60+ years of age) because of the highly conta-
gious nature of the infection. The general approach to treatment
includes the use of systemic antifungal drugs, with or without
topical treatment, together with environmental decontamination.
Lesions should be mapped and photographed before treatment is
initiated so that progress can be monitored over time.
Management of dermatophytosis in multicat households
can be frustrating, because (1) all in-contact cats must be
treated; (2) some cats are colonized and have no visible lesions;
and (3) testing and treatment require considerable energy,
time, monitoring, and expense. For group-housed animals, all
in-contact animals should be examined and cultured using the
toothbrush technique. Ideally all animals with positive cultures
(and definitely those with clinical signs) should be treated with
both systemic and topical antifungal therapy, whereas all unaf-
fected, culture-negative animals should be monitored for devel-
opment of lesions and treated with topical antifungal drugs
(ideally lime sulfur).
Topical Antifungal Drug Treatment
Topical antifungal drug treatment is not as effective as systemic
treatment, but inactivates fungal spores on the haircoat and
reduces environmental contamination. Spot treatments are not
recommended, because spores are distributed all over the hair-
coat even when skin lesions are localized. The two most effective
topical treatments are lime sulfur (1:16 dilution, or 237 mL in
3.6 L of water) or 0.2% enilconazole, both of which should be
applied twice weekly.42-44 Miconazole-chlorhexidine shampoos
are also an option but may not be as effective as lime sulfur.45,46
Lime sulfur shampoos, sprays, or dips have a foul odor and can
FIGURE 58-10  Histopathology that shows a cross-sectional view of a hair shaft
from a 3-year-old female spayed Boston terrier. Fungal hyphae are visible within the hair
shaft (arrows).

TABLE 58-3
Antimicrobial Drugs That Can Be Used for Systemic Treatment of Dermatophytosis*
Drug Dose (mg/kg)
Itraconazole† 5 PO
Fluconazole 5-10
Ketoconazole 10
Terbinafine† 30-40
Griseofulvin† (microsized) 25
Griseofulvin (ultramicrosized) 15
*See also Chapter 9 for more information on adverse reactions and drug administration before use.
†Administer itraconazole capsules, terbinafine, and griseofulvin with food. Reduce dose of itraconazole to 3 mg/kg q24h if using the suspension in cats.
stain light-colored haircoats but are safe for all age groups. A
reformulated lime sulfur product with an odor-masking agent
was not as effective in one clinical trial as a traditional lime
sulfur preparation.46 Lime sulfur products are most commonly
used in the United States but may not be readily available in
other parts of the world. Anecdotally, lime sulfur solutions have
been reported to cause oral ulcerations in cats, but these were
not detected in studies that evaluated cats that were allowed
to lick their coats after treatment.46,47 Ingestion of enilconazole
can cause hypersalivation, anorexia, and vomiting, and there
are rare anecdotal reports of severe adverse effects that include
death.11 Use of an Elizabethan collar is recommended to prevent
cats from licking topical solutions from the haircoat until the
treatment has dried. Chlorhexidine alone or povidone-iodine are
not sufficiently active against spores and are not recommended.
Whether or not the haircoat should be clipped before applica-
tion of these treatments is controversial. Clipping removes con-
taminated hair and facilitates penetration of topical treatments,
but may also serve to contaminate the environment and may
create or exacerbate skin lesions if clipping disrupts the skin. It
is recommended that clipping be used for longhaired cats and all
cats in an affected Persian or Himalayan cattery. Clippers must
be thoroughly disinfected with 10% bleach or concentrated
accelerated hydrogen peroxide solutions afterward.
Systemic Antifungal Drug Treatment
Systemic antifungal drug treatment decreases the duration
and severity of feline dermatophytosis (Table 58-3).42 How-
ever, it should be used in conjunction with topical treatment
and environmental decontamination when groups of animals
are affected. Antifungal drugs with activity against dermato-
phytes are griseofulvin, ketoconazole, fluconazole, itraconazole,
and terbinafine (see Chapter 9 for additional information on
these drugs). Itraconazole is the drug of choice for treatment
of dermatophytosis, because adverse reactions are uncommon
and it has equal or greater efficacy when compared with gris-
eofulvin.48 Fluconazole is less active than other azole antifungal
drugs against dermatophytes in  vitro (as is the case for other
fungi),49,50 but controlled clinical trials that compare the efficacy
of fluconazole to that of itraconazole for treatment of dermato-
phytosis are needed. The chitin inhibitor lufenuron (traditionally
administered orally to prevent flea infestations) has been evalu-
ated for treatment of dermatophytosis,51,52 but its efficacy has
been questioned and its use is no longer recommended.10,11,24
CHAPTER 58  Dermatophytosis 565
Route Interval (hours)
12-24
PO 12-24
PO 12
PO 24
PO 12
PO 12
Environmental Decontamination
In some situations, environmental decontamination is extremely
difficult or impossible because of the hardiness of dermatophyte
spores and their widespread dispersal on surfaces that cannot
be adequately disinfected. Even concentrated (1:10) bleach solu-
tions are not 100% active against dermatophyte spores; com-
plete disinfection requires undiluted household bleach or 1%
formalin. Both are too toxic and caustic for routine use.
All potentially contaminated beds and blankets should be
either discarded or washed with bleach through the use of a hot
cycle and a hot dryer. Leashes, collars, and grooming equipment
should be discarded, and carpets and furniture should be vacu-
umed to remove all visible hair and then steam cleaned. Because
vacuum cleaners are a source of recontamination, they may need
to be replaced after initial cleaning efforts; at the very least the
vacuum bag should be discarded. Disinfection of hard surfaces
should be performed with 1:10 to 1:100 bleach solution, 0.2%
enilconazole solution, or (where available) an enilconazole fog-
ger. Vehicles and carriers used to transport animals must also be
appropriately cleaned. According to label information, concen-
trated (1:16) accelerated hydrogen peroxide solutions may also
inactivate dermatophytes and reduce environmental contamina-
tion, but this requires further evaluation (see Chapter 11).
Duration of Treatment
The duration of treatment of dogs and cats with dermatophy-
tosis is based on serial physical examinations and importantly,
follow-up culture results. Usually, several months of treatment
are required. In general, animals should be examined and a cul-
ture performed at least monthly (and no more frequently than
weekly) during treatment. Treatment should be continued until
two successive cultures are negative and active lesions have
resolved. For group-housed animals, at least three successive
negative cultures are suggested. Some authors advocate once-
weekly cultures because this may permit earlier discontinua-
tion of treatment.11 If serial cultures are not possible because
of financial limitations, treatment should be continued for 2 to
4 weeks after complete resolution of clinical signs. Typically,
treatment is continued for at least 10 to 12 weeks.
Prognosis
The prognosis is good for resolution of dermatophytosis in
young animals, especially in households with only a single
animal or very low numbers of animals. Complicated disease
566 SECTION 3  Fungal and Algal Diseases

such as dermatophytic mycetomas are difficult to resolve and

require very long treatment durations; in some cats with myce-
tomas, surgery may ultimately be required for complete lesion
resolution. The prognosis for rapid resolution of dermatophy-
tosis may also be worse for animals with irreversible underlying
immunocompromise. Hair regrowth may not occur in regions
of scarring.

Immunity and Vaccination

Resolution of dermatophytosis depends on a delayed type-
hypersensitivity immune response, which involves the activity
of macrophages and IFN-γ.22 Less invasive dermatophytes may
evade the host immune response by limiting their presence to
the superficial corneal epithelial cells and hair shafts. A vaccine
for prevention of feline M. canis infections was licensed in the
United States in the mid-1990s but was not sufficiently effica-
cious and is no longer available.

Prevention
Prevention of dermatophytosis relies on avoidance of contact
with infected or carrier animals or fomites, but this may be
impossible in some situations. For scenarios that involve group-
housed animals, clean (culture-negative) animals, incoming ani-
mals, and animals undergoing treatment (culture-positive) must
be kept in separate rooms, and ideally in individual cages. Once
animals in the contaminated room are culture negative, they
are moved to the clean room, and the contaminated room is
thoroughly disinfected. Culture and topical treatment should be
considered for any incoming animals to a clean facility.
In veterinary hospitals, animals with known dermatophy-
tosis should be scheduled for examination late in the day and
should be carried directly from the parking lot to an appropri-
ate examination room that can subsequently be easily cleaned
(the animal should not wait in the reception area). Strict atten-
tion to barrier precautions is required (see Chapter 11). If hos-
pitalization is necessary, animals with suspected or documented
M. canis infections should be housed in isolation.
Public Health Aspects
Forms of human dermatophytosis include tinea capitis (scalp),
tinea pedis (“athlete’s foot”), tinea barbae (bearded area),
tinea cruris (“jock itch”), tinea corporis (body), tinea imbri-
cata (a variant of tinea corporis), tinea manuum (hand), tinea
faciei (face), and tinea unguium (nails).1 Dermatophyte spe-
cies that cause disease in dogs and cats have the potential to
cause human disease. Microsporum canis is the most common
zoophilic dermatophyte implicated. Zoophilic dermatophytes
are most often associated with tinea capitis or tinea corporis.
A high prevalence of infection with M. canis has been reported
in children that have tinea capitis (scalp infection) from con-
tinental Europe and Asia.53-55 M. canis is now a rare cause of
FIGURE 58-11  Boy with multiple lesions of dermatophytosis on the face and body.
The boy had juvenile rheumatoid arthritis. The boy’s cat had a lesion on the right side of its
face. (Courtesy Dr. Richard Malik.)
tinea capitis in the United States, with most cases caused by
anthropophilic dermatophytes such as T. tonsurans.56
Young children and the immunocompromised (e.g., trans-
plant recipients) are most susceptible to dermatophytosis and
may develop deep, recalcitrant, and/or disseminated lesions
as a result of contact with infected pets.57,58 Disease has been
described in veterinarians and veterinary students.11,35 In general,
zoophilic and geophilic species evoke a more profound inflamma-
tory response than anthropophilic dermatophytes; the latter tend
to cause more chronic infections in people.1 Typical “ringworm”
lesions in humans are erythematous, circular lesions with a raised
margin (Figure 58-11), but atypical forms that mimic other skin
diseases such as atopic dermatitis and lupus have been described.59
Humans who handle infected animals should wear protective
clothing and gloves. Young children or the immunocompromised
should avoid any contact with these animals. Toothbrush cul-
tures should be considered for cats that are adopted from shelters
or multicat households before they are introduced to households
where immunocompromised individuals reside.

CASE EXAMPLE
Signalment: “Jack”, a 4.5-year-old male neutered smooth-
coated fox terrier from northern California
History: Jack was evaluated for chronic diarrhea, a distended
abdomen, and loss of hair on the face. Jack’s owners adopted
him when he was 14 months old, at which time he had no
clinical signs of illness. At 2 years of age, the owners noted that
Jack began to rub his muzzle on objects. The intensity of this
behavior at times resulted in excoriations that would bleed.
Jack had been seen at another veterinary clinic where he was
treated with a diet that was “hypoallergenic.” The owners then
continued to try to feed a variety of different diets for the skin
problem, with a change to a new diet approximately every
3 months. The most recently fed diets were a prescription
intestinal diet and a prescription venison and potato diet. Diet
trials were never strict elimination trials, because the dog was
fed the same treats throughout the diet trial process. There
was no improvement in the skin lesions, which progressed to
cause alopecia that involved the entire aspect of his face (see
Figure 58-1, A). Approximately 18 months before Jack was
evaluated, intermittent diarrhea had developed. The diarrhea
was brown, occurred once or twice a day, and was sometimes
accompanied by tenesmus. It occasionally contained
mucus but never blood, and varied from soft to watery in
consistency. In addition to this history, over the preceding
2 years, Jack had been treated intermittently with prednisone
(0.5 to 0.75 mg/kg PO q12h) for the pruritus. The prednisone
never improved the skin condition but was occasionally
associated with resolution of the diarrhea. Other medications
used at various time points over the previous 2 years included
hydroxyzine, metronidazole, tylosin, cephalexin, and a topical
otic preparation that contained thiabendazole, neomycin,
and dexamethasone. Although Jack’s appetite was generally
good, he was described as a picky eater and had intermittent
lethargy when his diarrhea was severe. The owners reported
that over the most recent few weeks he had lost weight and
muscle mass and had developed a distended abdomen. Jack
lived on a ranch with multiple other dogs (the owner bred
poodles and shelties), cats, and horses, none of which were
unwell or had skin lesions. He spent most of his time outdoors
on the farm and loved to dig in rodent holes.
Current medications: Prednisone, 1.4 mg/kg PO q12h
Physical Examination:
Body Weight: 7.1 kg.
General: Bright, alert, responsive, hydrated. T = 100.3°F
(37.9°C), HR = 120 beats/min, RR = 40 breaths/min, mucous
membranes pink/pigmented, CRT <2 s.
Integument: A dull, unkempt haircoat was noted. An area of
partial alopecia with crusts was present over the right caudal
tarsus. Diffuse, complete alopecia with serocellular and
hemorrhagic crusting and multifocal, discrete ulcerations
was present over the entire dorsal muzzle (see Figure 58-1, A).
The skin within the alopecic area was hyperpigmented and
had a shiny appearance compatible with scarring. Periocular
alopecia with crusts was also present bilaterally and was
confluent with the erythematous, advancing edge of the
facial lesion. The skin on the ventral abdomen was thin with
prominent subcutaneous vessels.
CHAPTER 58  Dermatophytosis 567
Eyes, Ears, and Throat: No clinically significant abnormalities
of the eyes or ears were noted. On oral examination, the
dentition was severely worn, and there was mild to moderate
periodontal disease.
Musculoskeletal: Normal ambulation. Body condition score
was 3/9.
Cardiovascular and Respiratory Systems: No clinically
significant abnormalities were noted.
Gastrointestinal and Genitourinary: The abdomen was
moderately distended with a palpable fluid wave. Rectal
examination revealed only a moderate amount of formed
but soft feces.
Lymph Nodes: No peripheral lymphadenopathy was detected.
Laboratory Findings:
CBC:
HCT 39.8% (40%-55%)
MCV 69.7 fL (65-75 fL)
MCHC 34.2 g/dL (33-36 g/dL)
WBC 18,810 cells/µL (6000-13,000 cells/µL)
Neutrophils 16,177 cells/µL (3000-10,500 cells/µL)
Band neutrophils 188 cells/µL
Lymphocytes 376 cells/µL (1000-4000 cells/µL)
Monocytes 1693 cells/µL (150-1200 cells/µL)
Eosinophils 376 cells/µL (0-1500 cells/µL)
Platelets clumped, rare nucleated RBC/100 WBC.
Serum Chemistry Profile:
Sodium 144 mmol/L (145-154 mmol/L)
Potassium 4.1 mmol/L (3.6-5.3 mmol/L)
Chloride 115 mmol/L (108-118 mmol/L)
Bicarbonate 24 mmol/L (16-26 mmol/L)
Phosphorus 1.9 mg/dL (3.0-6.2 mg/dL)
Calcium 6.6 mg/dL (9.7-11.5 mg/dL)
BUN 10 mg/dL (5-21 mg/dL)
Creatinine 0.5 mg/dL (0.3-1.2 mg/dL)
Glucose 108 mg/dL (64-123 mg/dL)
Total protein 2.9 g/dL (5.4-7.6 g/dL)
Albumin 1.5 g/dL (3.0-4.4 g/dL)
Globulin 1.4 g/dL (1.8-3.9 g/dL)
ALT 85 U/L (19-67 U/L)
AST 109 U/L (19-42 U/L)
ALP 23 U/L (21-170 U/L)
Creatine kinase 309 U/L (51-399 U/L)
GGT < 3 U/L (0-6 U/L)
Cholesterol 88 mg/dL (135-361 mg/dL)
Total bilirubin < 0.1 mg/dL (0-0.2 mg/dL)
Magnesium 1.8 mg/dL (1.5-2.6 mg/dL).
Serum Vitamin B12 and Folate: Vitamin B12 314 ng/L (272-875
ng/L), folate 9.1 ng/mL (6.5-18.6 ng/mL).
Serum Ionized Magnesium Concentration: 0.44 mmol/L
(0.40-0.52 mmol/L).
Urinalysis (Cystocentesis): SGr 1.018; pH 6.5, no protein,
bilirubin, hemoprotein, or glucose, 0-2 WBC/HPF, no RBC/
HPF, no crystals or bacteria, rare granular casts, few lipid
droplets.
Imaging Findings:
Thoracic Radiographs: No abnormalities were detected in
the thorax. In the portion of visible abdomen, there was
decreased serosal detail.
Abdominal Ultrasound: There was marked thickening of
the mucosal layer of the small intestine diffusely, with
568 SECTION 3  Fungal and Algal Diseases
hyperechoic striations. A moderate volume of anechoic
peritoneal effusion was present.
Cytologic Findings: Abdominal effusion: the fluid was
colorless with a total protein of 0.3 g/dL, low numbers of
erythrocytes, and 380 nucleated cells/µL (38% neutrophils,
2% small mononuclear cells, and 60% large mononuclear
cells). Nucleated cells were a mixture of nondegenerate
neutrophils and mononuclear cells of normal morphology.
One large cluster of reactive mesothelial cells was observed.
The fluid was interpreted as a transudate.
Microbiologic Testing: Skin scrapings and hair pluck
cytology (trichogram) (from face and tarsus): Large numbers
of dermatophyte hyphae and spores were present. No mites
were seen.
Fungal culture (hair specimens from periphery of lesions on
face and tarsus): Small numbers of Trichophyton mentagro-
phytes grew after 4 days.
Diagnosis: Protein-losing enteropathy (suspect lymphangiec-
tasia), likely iatrogenic hyperadrenocorticism, and dermato-
phytosis due to T. mentagrophytes infection.
Treatment: Before the results of the fungal culture became
available, fluconazole (7 mg/kg PO q24h) was prescribed
based on the high index of suspicion from cytologic
identification of fungal elements, together with an oatmeal
shampoo to be used twice weekly. The dose of prednisone
was reduced to 0.5 mg/kg PO q12h and strict instructions
were given to feed a prescription low-fat diet and no other
foods (including treats). At a recheck 3 weeks later, the
owners reported that Jack was doing very well. His diarrhea,
abdominal distention, and facial pruritus had resolved;
he was more energetic; and his appetite had improved.
SUGGESTED READINGS
Chermette R, Ferreiro L, Guillot J. Dermatophytoses in animals. Myco-
pathologia. 2008;166:385-405.
Moriello KA. Treatment of dermatophytosis in dogs and cats: review of
published studies. Vet Dermatol. 2004;15:99-107.
Vermout S, Tabart J, Baldo A, et al. Pathogenesis of dermatophytosis.
Mycopathologia. 2008;166:267-275.
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Comments: Persistent dermatophytosis in this dog was
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15. Terreni AA, Gregg Jr WB, Morris PR, et al. Epidermophyton floc-
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19. Moriello KA, Kunkle G, DeBoer DJ. Isolation of dermatophytes
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two different geographic regions of the United States. Vet Derma-
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20. Moriello KA, Deboer DJ. Fungal flora of the haircoat of cats with
and without dermatophytosis. J Med Vet Mycol. 1991;29:285-292.
21. Reche Jr A, Daniel AG, Lazaro Strauss TC, et al. Cutaneous myco-
flora and CD4:CD8 ratio of cats infected with feline immunodefi-
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22. Vermout S, Tabart J, Baldo A, et al. Pathogenesis of dermatophyto-
sis. Mycopathologia. 2008;166:267-275.
23. Sriranganadane D, Waridel P, Salamin K, et  al. Identification of
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25. Cornegliani L, Persico P, Colombo S. Canine nodular dermatophy-
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28. Nobre Mde O, Negri Mueller E, Teixeira Tillmann M, et al. Dis-
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29. Bergman RL, Medleau L, Hnilica K, et al. Dermatophyte granulo-
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30. Chang SC, Liao JW, Shyu CL, et al. Dermatophytic pseudomyceto-
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31. Bond R, Pocknell AM, Tozet CE. Pseudomycetoma caused by
Microsporum canis in a Persian cat: lack of response to oral terbin-
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33. Chermette R, Bussieras S, Jeanmonod P, et  al. Dermatophytie à
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34. Guillot J, Latie L, Deville M, et al. Evaluation of the dermatophyte
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35. Hermoso de Mendoza M, Hermoso de Mendoza J, Alonso JM, et al.
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36. Nenoff P, Erhard M, Simon JC, et al. MALDI-TOF mass spectrom-
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37. Cafarchia C, Gasser RB, Figueredo LA, et al. An improved molecu-
lar diagnostic assay for canine and feline dermatophytosis. Med
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38. Nardoni S, Franceschi A, Mancianti F. Identification of Micros-
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embedded veterinary specimens using a common PCR protocol.
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39. Yuksel T, Ilkit M. Identification of rare macroconidia-producing der-
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41. Peters J, Scott DW, Erb HN, et al. Comparative analysis of canine
dermatophytosis and superficial pemphigus for the prevalence of
dermatophytes and acantholytic keratinocytes: a histopathological
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42. Moriello KA. Treatment of dermatophytosis in dogs and cats:
review of published studies. Vet Dermatol. 2004;15:99-107.
43. Diesel A, Verbrugge M, Moriello KA. Efficacy of eight commer-
cial formulations of lime sulphur on in vitro growth inhibition of
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46. Newbury S, Moriello KA, Kwochka KW, et al. Use of itraconazole
and either lime sulphur or Malaseb Concentrate Rinse (R) to treat
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47. Newbury S, Moriello K, Verbrugge M, et al. Use of lime sulphur
and itraconazole to treat shelter cats naturally infected with Micros-
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48. Moriello KA, DeBoer DJ. Efficacy of griseofulvin and itraconazole
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50. Mota CR, Miranda KC, Lemos Jde A, et al. Comparison of in vitro
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51. Guillot J, Malandain E, Jankowski F, et al. Evaluation of the efficacy
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Pediatr Dermatol. 2012;29:409-413.
CHAPTER 59
Malassezia Infections
Jane E. Sykes, Terry M. Nagle, and Stephen D. White
Overview of Malassezia Dermatitis
First Described: 1874, in France, by Louis-Charles Malassez
(the fungus was initially named Pityrosporum ovale)1
Cause: Malassezia pachydermatis; less commonly, lipid-
dependent species such as Malassezia globosa, Malasse-
zia sympodialis, Malassezia nana, Malassezia slooffiae, and
Malassezia furfur are involved.
Affected Hosts: Dogs and to a lesser extent cats
Geographic Distribution: Worldwide
Mode of Transmission: Direct contact and opportunistic pro-
liferation of commensal organisms
Major Clinical Signs: Cutaneous erythema, pruritus, alopecia,
scaling, greasy exudation, hyperpigmentation, lichenifi-
cation, malodor
Differential Diagnoses: Dermatophytosis, demodicosis, aller-
gic dermatitis, drug eruptions, pyoderma
Human Health Significance: M. pachydermatis does not nor-
mally colonize human skin but can rarely infect immuno-
suppressed adults and cause catheter-related infections
in neonatal intensive care units.
Etiology and Epidemiology
Malassezia spp. (formerly Pityrosporum spp.) are lipophilic
yeasts that normally colonize animal and human skin in low
numbers. At least 13 species of Malassezia exist. The vast major-
ity of species are classified as lipid dependent: when grown in
the laboratory, these species have an absolute requirement for
long-chain fatty acids, which are used as a source of carbon.
In contrast, Malassezia pachydermatis, the most commonly
isolated yeast species from the skin and ears of healthy dogs
and cats, is non–lipid dependent when grown in the laboratory.
Lipid-dependent species, including Malassezia globosa, Malas-
sezia sympodialis, Malassezia nana, Malassezia slooffiae, and
Malassezia furfur, can colonize the skin of healthy cats.2-5
In healthy dogs, Malassezia spp. are most commonly found
in the ear canals, on the lips, axillae, interdigital spaces, anal
sacs, and occasionally the nose and vagina. It has been suggested
that a symbiotic relationship exists between Malassezia spp. and
commensal staphylococci; each maintains a local microenviron-
ment that benefits the other.
Malassezia spp. are common opportunistic contributors to
chronic dermatitis and otitis externa in dogs, and to a lesser
extent in cats. They are isolated more frequently from the
570
ears of dogs and cats with otitis externa than from the ears of
healthy dogs and cats.6,7 Dogs with skin disease can have 100-
to 10,000-fold increases in skin population densities of Malas-
sezia spp. compared with healthy dogs.8 Species and strains of
Malassezia vary in virulence and tropism for different anatomic
sites and host species.9,10 A variety of lipid-dependent Malasse-
zia spp. occasionally have been isolated from dogs and cats with
otitis externa.6,7,11-14
Malassezia spp. infection can occur in dogs and cats of any
age, breed, and sex, but some dog breeds are thought to be pre-
disposed, including American cocker spaniels, West Highland
white terriers, basset hounds, poodles, and Australian silky terri-
ers.15 Sphynx and Devon Rex cats have high rates of Malassezia
spp. colonization when compared with domestic shorthair cats.16
Clinical Features
Signs and Their Pathogenesis
Malassezia spp. proliferate opportunistically, becoming patho-
genic with alterations in host defenses or the skin surface
microclimate. Examples of underlying diseases include allergic
dermatitis, endocrinopathies, intertrigo, primary keratinization/
cornification disorders, and, in cats, underlying neoplasia or ret-
rovirus infections.17,18 A history of treatment with antibiotics
was a predisposing factor in one study.19 Prolonged glucocor-
ticoid treatment can also lead to yeast proliferation. The yeasts
adhere to the cells of the stratum corneum and secrete lipases,
proteinases, phospholipases, and acid sphingomyelinases.20
The resulting skin lesions may be localized or generalized, are
thought to result from inflammation, and, in some animals,
hypersensitivity reactions to yeast antigens.21,22 The more ery-
thematous the interdigital region is in an atopic dog, the more
likely a Malassezia spp. infection is present.23
The most commonly affected anatomic sites are the ear
canals, skin folds (including the periocular and perioral skin,
ventral neck, axillae, inguinal regions, and perineum), and
interdigital skin (Figure 59-1). Clinical signs include ery-
thema, pruritus, alopecia, scaling, and a greasy exudate. The
claw beds may develop a reddish-brown stain. Chronicity
results in hyperpigmentation, lichenification, and stenosis of
the ear canal, and a pungent, offensive odor develops. Pruritus
may result in excoriations. Devon Rex and Sphynx cats can
develop a greasy dermatitis associated with increased numbers
of Malassezia spp., which can respond dramatically to anti-
fungal drug treatment.8
Physical Examination
A thorough physical and dermatologic examination aids diag-
nosis of Malassezia spp. infection and provides important clues
 CHAPTER 59  Malassezia Infections 571

A B
FIGURE 59-1  Five-year-old male neutered pug with a 1 year history of otitis externa and generalized pruritus. Malassezia dermatitis and bacterial pyoderma secondary to underlying
atopic dermatitis was diagnosed. Note the distribution of lesions, which affect the facial folds and ventral neck (A) and axillae (B).

as to the underlying disorder leading to opportunistic prolifera-

tion of Malassezia spp. It also allows identification of lesions
consistent with concurrent pyoderma (see Chapter 84).

Diagnosis
Diagnosis of Malassezia spp. dermatitis is based on clinical signs,
findings on cytologic examination, and a positive response to
antifungal drug treatment.

Microbiologic Testing
Skin Scrapings
Skin scrapings are important in animals suspected to have Mal-
assezia spp. dermatitis in order to rule out demodicosis, which
can mimic Malassezia spp. dermatitis.

Cytologic Examination
Cytology is an important tool for diagnosis and is generally
performed using clear (not frosted) acetate (Scotch) tape prepa-
rations of the skin. The tape is pressed to the skin, dipped in
the final step basophilic (blue) Diff-Quik solution, rinsed, and
applied to a slide on which a drop of immersion oil has been
placed. Alternatively, the tape is pressed against the affected
skin several times and then placed on a dry glass slide, and a
small amount of the basophilic Diff-Quik solution is injected
under the tape. The tape is then examined using a light micro-
scope, with an additional drop of immersion oil to allow
identification of bacteria and Malassezia spp. using the 100˜
objective. The yeasts stain deeply basophilic and exhibit wide-
based budding, resembling “footprints,” “peanuts,” or “snow-
men” (Figure 59-2). An inflammatory cellular response is often
not present, even when Malassezia spp. contribute to disease
progression.
Estimation of yeast counts, although only semiquantitative,
may help to determine the role of Malassezia in otitis and der-
matitis, but there are no clear guidelines as to what constitutes a
normal yeast population size. In addition, yeast counts do not take
FIGURE 59-2  Tape preparation from the skin of a dog with Malassezia dermatitis.
Note broad-based budding yeasts, which have the appearance of a footprint. Modified
Wright’s stain, 1000× oil magnification.
into account other factors, such as strain variation in virulence
or host hypersensitivity.10,24 For Malassezia spp. dermatitis, mean
counts of 1 or more yeast per oil immersion field (1000× magnifi-
cation) are considered abnormal in dogs.25 For ears, a mean yeast
count of approximately 5 or more organisms per oil immersion
field has been suggested as abnormal.25 Cats may be colonized
with higher numbers of yeasts, and 12 or more organisms per
high dry field has been suggested as abnormal in feline ear canals
in another study.26 The ear canals of healthy dogs and cats have
mean counts of 2 or fewer yeasts per high dry field (400× magnifi-
cation), and intermediate counts represent a gray zone.26
Fungal Culture
Fungal culture and susceptibility testing for Malassezia spp.
is not routinely performed for clinical diagnostic purposes,
572 SECTION 3  Fungal and Algal Diseases
TABLE 59-1
Systemic Antifungal Drugs Used for Treatment of Malassezia Dermatitis and Otitis Externa in Dogs
Drug Dose (mg/kg) Route
Ketoconazole 5 to 10 PO
Itraconazole 5* PO
Fluconazole 5 PO
Terbinafine 30 PO
*Dose applies to the capsules. Reduce dose to 3 mg/kg if using the suspension.
although methods that use broth dilution or E-testing for sus-
ceptibility testing have been described. The organism can be
cultured from a tape preparation, which in the laboratory is
mounted on a drop of olive oil that has been placed on fungal
isolation media.25 Growth occurs within 7 days of incubation.
Currently, most Malassezia spp. infections are broadly suscep-
tible and respond well to treatment of the yeast infection and
any underlying disorders present. One azole-resistant isolate of
M. pachydermatis was identified in a dog with dermatitis, but
this was not associated with resistance in vivo.27
Treatment and Prognosis
Treatment of Malassezia spp. dermatitis and otitis requires iden-
tification and, if possible, treatment of the underlying cause, in
addition to antifungal drug treatment. Because Malassezia spp.
infection is superficial, topical treatments may be adequate to
resolve infection. A variety of topical otic preparations are avail-
able for treatment of Malassezia otitis. These contain antifun-
gals such as clotrimazole, miconazole, or posaconazole, which
are generally combined with a glucocorticoid and an antibacte-
rial (see Chapter 84). There is good evidence that Malassezia
dermatitis can be successfully treated with twice-weekly sham-
pooing with a 2% miconazole/2% chlorhexidine shampoo (e.g.,
Malaseb, DVM Pharmaceuticals) for 3 weeks,24 and this can
also prevent recurrence. A 3% chlorhexidine shampoo may be
equally efficacious.28 Systemic antifungals that have been shown
to have efficacy for treatment of Malassezia infections include
ketoconazole, itraconazole, fluconazole, and terbinafine, which
are generally administered for 3 weeks (Table 59-1).29,30 Longer
periods of treatment may be required for severe infections or
infections of the claw bed.8 Administration of terbinafine on
two consecutive days each week (i.e., in a pulsatile fashion)
may be as effective as daily administration, but more studies
are required.31 Treatment should be monitored through serial
dermatologic examinations and cytologic evaluation of tape
preparations. The prognosis for resolution depends on the abil-
ity to identify and address the underlying cause for proliferation
of Malassezia spp.
Public Health Aspects
In human beings, Malassezia infections most commonly result
from infection by M. furfur. This organism belongs to the
normal human cutaneous flora and proliferates opportunisti-
cally following immunocompromise. Yeast proliferation can
lead to Malassezia folliculitis, seborrheic dermatitis, catheter-
related fungemia, and a variety of other invasive infections.32
Interval (hours) Duration (days)
12 3 to 4 weeks or until
24 cytologic resolution
24
24
Administration of lipid-containing total parenteral nutrition
solutions is a risk factor for disseminated infections in humans,
which often occur in neonates.33,34
M. pachydermatis is not part of the normal human cutane-
ous microflora. Human disease associated with M. pachyder-
matis infection occurs rarely. Nosocomial infections with M.
pachydermatis have been reported in neonates in intensive care
units.35 One epidemic was associated with colonization of health
care workers’ pet dogs with a strain identical to that causing
disease, as determined by molecular typing with pulsed-field gel
electrophoresis.36 Cutaneous infections have also been reported
in adults with underlying immunosuppressive disease.30 Owners
of dogs with atopic skin disease were 11 times more likely to
carry M. pachydermatis on their hands than owners of healthy
dogs, as determined using culture (39% vs. 6%).25 The owners
of both groups of dogs were equally likely to carry the yeast on
their hands as determined by PCR testing (93% and 94%). It
was concluded that mechanical carriage of the organism by dog
owners appears to be of low risk to human health, given how
rarely disease due to M. pachydermatis occurs. Hand washing
is likely to reduce the rate of carriage of M. pachydermatis, but
this has not been well studied.
CASE EXAMPLE
See case example in Chapter 84.
SUGGESTED READING
Tragiannidis A, Bisping G, Koehler G, et al. Minireview: Malassezia infec-
tions in immunocompromised patients. Mycoses. 2010;53(3):187-195.
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3. Bond R, Howell SA, Haywood PJ, et  al. Isolation of Malassezia
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4. Crespo MJ, Abarca ML, Cabañes FJ. Isolation of Malassezia furfur
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5. Volk AV, Belyavin CE, Varjonen K, et al. Malassezia pachydermatis
and M. nana predominate amongst the cutaneous mycobiotia of
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6. Cafarchia C, Gallo S, Capelli G, et al. Occurrence and population
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7. Dizotti CE, Coutinho SD. Isolation of Malassezia pachydermatis
and M. sympodialis from the external ear canal of cats with and
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8. Bond R. Superficial veterinary mycoses. Clin Dermatol. 2010;28(2):
226-236.
9. Duarte ER, Hamdan JS. RAPD differentiation of Malassezia spp.
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11. Cafarchia C, Latrofa MS, Figueredo LA, et  al. Physiological and
molecular characterization of atypical lipid-dependent Malassezia
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13. Crespo MJ, Abarca ML, Cabaûes FJ. Atypical lipid-dependent
Malassezia species isolated from dogs with otitis externa. J Clin
Microbiol. 2000;38(6):2383-2385.
14. Shokri H, Khosravi A, Rad M, et al. Occurrence of Malassezia spe-
cies in Persian and domestic short hair cats with and without otitis
externa. J Vet Med Sci. 2010;72(3):293-296.
15. Scott DW, Miller WH, Griffin CE. Bacterial skin diseases. In: Muller
and Kirk’s Small Animal Dermatology. 6th ed. Philadelphia, PA:
WB Saunders; 2001:274-335.
16. Volk AV, Belyavin CE, Varjonen K, et al. Malassezia pachyderma-
tis and M. nana predominate amongst the cutaneous mycobiota of
Sphynx cats. J Fel Med Surg. 2010;2(12):917-922.
17. Sierra P, Guillot J, Jacob H, et al. Fungal flora on cutaneous and
mucosal surfaces of cats infected with feline immunodeficiency
virus or feline leukemia virus. Am J Vet Res. 2000;61(2):158-161.
18. Perrins N, Gaudiano F, Bond R. Carriage of Malassezia spp. yeasts
in cats with diabetes mellitus, hyperthyroidism and neoplasia. Med
Mycol. 2007;45(6):541-546.
19. Plant JD, Rosenkrantz WS, Griffin CE. Factors associated with and
prevalence of high Malassezia pachydermatis numbers on dog skin.
J Am Vet Med Assoc. 1992;201(6):879-882.
20. Coutinho SD, Paula CR. Proteinase, phospholipase, hyaluronidase
and chondroitin-sulphatase production by Malassezia pachyderma-
tis. Med Mycol. 2000;38(1):73-76.
21. Bond R, Curtis CF, Hendricks A, et  al. Intradermal test reac-
tivity to Malassezia pachydermatis in atopic dogs. Vet Rec.
2002;150(14):448-449.
22. Kim HJ, Kim ET, Lim CY, et al. The immunoglobulin G response
to Malassezia pachydermatis extracts in atopic and non-atopic
dogs. Can Vet J. 2010;51(8):869-872.
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23. White SD, Bourdeau P, Blumstein P, et al. Comparison via cytology
and culture of carriage of Malassezia pachydermatis in atopic and
healthy dogs. In: Kwochka KW, Willemse T, von Tscharner C, eds.
Advances in Veterinary Dermatology. vol. 3. Oxford, UK: Butter-
worth Heinemann; 1998:292-298.
24. Negre A, Bensignor E, Guillot J. Evidence-based veterinary derma-
tology: a systematic review of interventions for Malassezia derma-
titis in dogs. Vet Dermatol. 2009;20(1):1-12.
25. Morris DO. Malassezia pachydermatis carriage in dog owners.
Emerg Infect Dis. 2005;11(1):83-88.
26. Ginel PJ, Lucena R, Rodriguez JC, et  al. A semiquantitative
cytological evaluation of normal and pathological samples
from the external ear canal of dogs and cats. Vet Dermatol.
2002;13(3):151-156.
27. Nijima M, Kano R, Nagata M, et al. An azole-resistant isolate of
Malassezia pachydermatis. Vet Microbiol. 2011;149:288-290.
28. Maynard L, Rème CA, Viaud S. Comparison of two shampoos for
the treatment of Malassezia dermatitis: a randomised controlled
trial. J Small Anim Pract. 2011;52:566-572.
29. Rosales MS, Marsella R, Kunkle G, et al. Comparison of the clini-
cal efficacy of oral terbinafine and ketoconazole combined with
cephalexin in the treatment of Malassezia dermatitis in dogs—a
pilot study. Vet Dermatol. 2005;16(3):171-176.
30. Sickafoose L, Hosgood G, Snook T, et al. A noninferiority clinical
trial comparing fluconazole and ketoconazole in combination with
cephalexin for the treatment of dogs with Malassezia dermatitis.
Vet Ther. 2010;11(2):E1-E13.
31. Berger DJ, Lewis TP, Schick AE, et al. Comparison of once-daily
versus twice-weekly terbinafine administration for the treatment
of canine Malassezia dermatitis – a pilot study. Vet Dermatol.
2012;23:418–e79.
32. Tragiannidis A, Bisping G, Koehler G, et  al. Minireview: Mal-
assezia infections in immunocompromised patients. Mycoses.
2010;53(3):187-195.
33. Dankner WM, Spector SA, Fierer J, et al. Malassezia fungemia in
neonates and adults: complication of hyperalimentation. Rev Infect
Dis. 1987;9(4):743-753.
34. Chryssanthou E, Broberger U, Petrini B. Malassezia pachyder-
matis fungaemia in a neonatal intensive care unit. Acta Paediatr.
2001;90(3):323-327.
35. Larocco M, Dorenbaum A, Robinson A, et  al. Recovery of Mal-
assezia pachydermatis from eight infants in a neonatal intensive
care nursery: clinical and laboratory features. Pediatr Infect Dis J.
1988;7(6):398-401.
36. Chang HJ, Miller HL, Watkins N, et  al. An epidemic of Malas-
sezia pachydermatis in an intensive care nursery associated with
colonization of health care workers’ pet dogs. N Engl J Med.
1998;338(11):706-711.
CHAPTER 60
Blastomycosis
Jane E. Sykes and Lindsay K. Merkel
Overview of Blastomycosis
First Described: 1894, in the United States by Thomas Casper
Gilchrist.1 The organism was initially mistaken for a proto-
zoan parasite.
Cause: Blastomyces dermatitidis (an ascomycete, teleomorph
Ajellomyces dermatitidis)
Affected Hosts: Primarily humans and dogs, but also other
mammalian species that include cats, horses, ferrets, and
sea lions
Geographic Distribution: Primarily North America, especially
the south-central and upper midwestern states of the
United States; Canadian provinces that border the Great
Lakes; and a small area of the northeastern United States
and southeastern Canada near the St. Lawrence river
Mode of Transmission: Inhalation of conidia from the envi-
ronment; rarely cutaneous inoculation
Major Clinical Signs: Fever, inappetence, weight loss, cough,
tachypnea, respiratory difficulty, nodular or ulcerative
cutaneous lesions, ocular lesions (uveitis, chorioretinitis,
panophthalmitis), lameness, neurologic signs
Differential Diagnoses: Neoplasia, other deep mycoses, pro-
tothecosis, mycobacterial infections, systemic protozoal
infections (leishmaniasis, toxoplasmosis, neosporosis),
aspiration or foreign body pneumonia
Human Health Significance: B. dermatitidis causes disease in
humans, but direct transmission from animals to humans
does not occur (with the exception of rare bite wound
transmission from infected dogs). Dogs are a sentinel for
human exposure.
Etiology and Epidemiology
Blastomyces dermatitidis is a dimorphic fungus that grows as a
mycelial form in the environment and as a thick-walled budding
yeast in tissues (at 37°C). Hyphae within soil produce conidia
that are 2 to 10 µm in diameter. The conidia are thought to
be aerosolized and inhaled by the host, where they transform
into yeasts and produce a localized pulmonary or disseminated
disease known as blastomycosis (Figure 60-1). The organism’s
name (dermatitidis) stems from the fact that the skin is a com-
mon site to which the organism disseminates.
B. dermatitidis is primarily found in the eastern parts of
the United States, especially around the Great Lakes, along
574
the Ohio and Mississippi river valleys, and in the southeastern
states (Figure 60-2). A smaller focus of endemicity exists in the
St. Lawrence river region in the northeast, which extends up
into Ontario, Canada. Disease may also be seen in parts of Can-
ada around the Great Lakes and has also been reported from
Saskatchewan.2,3 Occasionally disease appears in nontraveled
dogs that reside in non-endemic parts of the United States, such
as in Wyoming and South Dakota. Canine blastomycosis has
also been reported from India.4 Human blastomycosis occurs in
Africa, but a different serotype is involved. Interestingly, reports
of canine disease from Africa are absent from the literature.
Blastomycosis was described in Europe in a dog with a travel
history to the United States.5
The ecologic niche of B. dermatitidis appears to be warm, moist,
sandy soil that is rich in organic debris such as decaying vegeta-
tion. However, this has not been thoroughly defined because the
fungus is difficult to isolate from the environment. No consistent
seasonality has been linked to disease. Genetic differences have
been reported among isolates of B. dermatitidis within the United
States. Using PCR-based typing methods, several different geno-
types have been described (A, B, C, D, and E).6 Analysis by mic-
rosatellite typing suggests the existence of at least two genetically
distinct groups, group 1 and group 2, the geographic distributions
of which overlap.7 In human patients, group 2 isolates may be
more likely to be associated with disease in older patients with
comorbid conditions, and group 2 isolates are also more likely to
be associated with dissemination than group 1 isolates.8
Young adult, large-breed (>15 kg) dogs are predisposed
to blastomycosis, and a slight male predisposition has been
Lung
Blood
FIGURE 60-1  Life cycle of Blastomyces dermatitidis. From the lung, the organism
can disseminate to a variety of tissues, but especially the eye, lymph nodes, skin, and
bones.
 CHAPTER 60  Blastomycosis 575

TABLE 60-1
Physical Examination Findings in 115 Dogs with Blastomycosis9
Clinical Sign Percent of Dogs
Fever 62
Lymphadenopathy 56
Harsh lung sounds 50
Skin lesions 49
Chorioretinitis 43
Anterior uveitis 42
Cough 32
FIGURE 60-2  Approximate geographic distribution of Blastomyces dermatitidis in the
United States. Emaciation 25
Retinal detachment 23
Cutaneous mass 16
Glaucoma 16
identified in some studies.9 Overrepresented breeds include
coonhounds, pointers, Weimaraners, Labrador retrievers, Tachypnea 16
golden retrievers, and Doberman pinschers.9,10 This may reflect Dehydration 15
the increased likelihood of exposure of these dog types to organ- Bony mass/swelling 14
isms in the environment, because they are more likely to be used
Nasal discharge  8
in outdoor activities such as hunting. Dogs younger than 6
months of age and as old as 17 years may be affected, but dogs in Neurologic signs  6
the 2- to 4-year age group have the highest risk of infection with Prostatomegaly  5
the 4- to 6-year age group a close second.9,10 Residence less than Mammary mass  3
400 m from a body of water increased the risk of blastomycosis
Orchitis  2
by a factor of 10 in one study from Louisiana,10 and exposure to
sites of soil disturbance or excavation may also increase the risk Synovial effusion  2
of infection. In one report, B. dermatitidis was isolated from a
woodpile near the Wisconsin River; over 14 years, 4 of 9 dogs
housed in a kennel close to the woodpile developed blastomyco-
sis.11 Most affected dogs are immunocompetent. Blastomycosis virtually any tissue in the body may be affected, and infection
is rarely reported in cats from endemic regions of the United has been reported in the mammary glands, nasal or oral cavities,
States and, as with other soil-borne mycoses such as histoplas- cardiac tissues (myocardium, endocardium, and pericardium),
mosis and cryptococcosis, blastomycosis can occur in cats that and very rarely in the kidneys, liver, spleen, peritoneum, gastro-
have been housed entirely indoors.12,13 In one study, 10% of 41 intestinal tract, and urinary bladder.20
affected cats tested positive for FeLV antigen.14 Clinical signs are variable. Dogs may develop subclini-
cal infections, acute or chronic disease that is localized to the
Clinical Features lungs and associated lymph nodes, or severe and progressive
signs that result from dissemination of the organism to multiple
Signs and Their Pathogenesis extrapulmonary sites. The incubation period is variable and not
Infection most often results from inhalation of conidia in the precisely known, but is estimated to range from 5 to 12 weeks.
environment. Direct inoculation of organisms followed by Nonspecific signs of illness such as lethargy, weakness, fever,
localized cutaneous disease and/or osteomyelitis (inoculation inappetence, and weight loss are common (Table 60-1). Other
blastomycosis) occurs rarely in dogs and human patients.15,16 common clinical manifestations (between 20% and 50% of
Once within the host, the conidia transform into yeasts that can affected dogs) include signs of respiratory involvement (such as
resist destruction by neutrophils. The yeasts trigger a pyogranu- cough and increased respiratory rate), lymphadenopathy, ocular
lomatous inflammatory response. Important virulence factors manifestations, cutaneous lesions, and lameness.10,21 Lameness
include a cell surface glycoprotein known as BAD-1 (previously may result from fungal osteomyelitis, arthritis, or rarely, hyper-
known as WI-1), and other cell wall components such as trophic osteopathy.22 Gastrointestinal signs (vomiting, diarrhea,
α-1,3-glucan and possibly melanin.17,18 BAD-1 is an adhe- hematemesis, and melena); polyuria or polydipsia; mammary
sin that binds to host cell receptors on macrophages. It may gland masses; laryngeal masses; nasal discharge, sneezing and/
also contribute to the yeast’s ability to evade the host immune or epistaxis; testicular masses; dysuria or hematuria secondary
response by influencing cytokine secretion and impairing com- to prostatic involvement; cardiac arrhythmias; and neurologic
plement activation.17,19 In some animals, the organism spreads signs due to meningoencephalitis or ependymitis occur less fre-
hematogenously from the lungs to associated lymph nodes and quently.10,23-29 Neurologic involvement secondary to extension
other extrapulmonary sites. Extrapulmonary sites of predilec- of intranasal or retrobulbar Blastomyces granulomas through the
tion include the skin, eye, bone, reproductive tissues (prostate calvarium can also occur.25,29 Rarely, thromboembolic disease
and testes), and the central nervous system (CNS). However, has been reported.30
576 SECTION 3  Fungal and Algal Diseases

B C
FIGURE 60-3  Disseminated blastomycosis in a labrador retriever. Nodular cutaneous lesions are present on the muzzle (A). There are also multiple, ulcerated, and draining skin lesions
on the distal limbs (B) and digit (C). (Courtesy Dr. Sheila Torres, University of Minnesota.)

In cats, clinical signs of blastomycosis are similar to those

in dogs, although neurologic and gastrointestinal involvement
may be more prevalent.31

Physical Examination Findings

Physical examination abnormalities in dogs with blastomy-
cosis commonly include fever, thin body condition, dehydra-
tion, and signs of respiratory involvement. Fever is present in
approximately 50% of affected dogs and is usually low grade
(103°F to 104°F or 39.4°C to 40.0°C) but occasionally exceeds
106°F (41.1°C).20,27 Respiratory signs include cough, tachy-
pnea, increased respiratory effort, and increased lung sounds
on thoracic auscultation. Other common findings on physical
examination are firm cutaneous or subcutaneous masses, drain-
ing or ulcerated skin lesions, and peripheral lymphadenopathy.
Careful palpation of the entire skin surface can reveal small
skin lesions. Cutaneous and subcutaneous lesions are frequently
found on the trunk, limbs, and digits and can also involve the
muzzle (Figure 60-3). Lesions of the tongue, gingiva, or muco-
cutaneous junctions may also be present (Figure 60-4). Facial
swelling, exophthalmos, or facial deformity has been described
in some affected dogs.25,32 Dogs with nasal cavity involve-
ment can have stertorous respiration, decreased nasal airflow,
or nasal discharge. Ocular signs may be unilateral or bilateral
and include chorioretinitis (sometimes with retinal detach-
ment); lesions consistent with optic neuritis; endophthalmitis
or panophthalmitis; uveitis with aqueous flare, iris bombé, syn- FIGURE 60-4  Nodular and ulcerated lesions of the tongue in a dog with dissemi-
echia, and miosis; cataract formation; conjunctivitis; keratitis; nated blastomycosis. (Courtesy Dr. Sheila Torres, University of Minnesota.)

A B
C
FIGURE 60-5  Cutaneous lesions in a cat with disseminated blastomycosis that involved the footpads (A), interdigital folds (B), and lips (C). (Courtesy Dr. Sheila Torres, University
of Minnesota.)
and photophobia.33,34 Severe ocular involvement may result in
increased intraocular pressures and/or loss of vision. Dogs with
musculoskeletal involvement may be lame, have firm swellings
associated with long bones, or exhibit joint swelling, warmth,
and/or pain. Dogs with testicular blastomycosis may have scro-
tal swelling or palpable testicular masses. Neurologic signs are
present in fewer than 5% of affected dogs and can include sei-
zures, hypermetria, decreased placing reactions, tetraparesis,
circling, ataxia, blindness, decreased or absent menace and/
or pupillary light reflexes, nystagmus, and decreased facial
sensation.
Physical examination findings in cats with blastomycosis are
similar to those described in dogs. These include fever, thin body
condition, peripheral lymphadenopathy, tachypnea, increased
or decreased lung sounds, ocular abnormalities (chorioretinitis
with retinal detachment, uveitis, panophthalmitis, and second-
ary glaucoma), nodular or draining skin lesions (Figure 60-5),
and a variety of neurologic signs that include obtundation,
CHAPTER 60  Blastomycosis 577
ataxia, pelvic limb paresis, circling, hyperesthesia, decreased
placing reactions, and blindness.13,31,35-37
Diagnosis
A diagnosis of blastomycosis is usually suspected based on
the presence of suspicious clinicopathologic abnormalities in a
dog or cat from an endemic area. The diagnosis is usually con-
firmed through cytologic examination of fine needle aspirates
of affected tissues (especially skin lesions and lymph nodes),
impression smears of draining skin lesions, respiratory lavage
specimens (transtracheal washes or bronchoalveolar lavage), or
body fluids (especially CSF or ocular fluid, but also synovial
fluid or urine sediment). Other methods of diagnosis include
histopathology (e.g., of bone or tissue biopsies), fungal culture,
and PCR-based assays (Table 60-2). Serologic assays that detect
antigen and antibody are also available, but when used alone,
these do not confirm a diagnosis of blastomycosis.
578 SECTION 3  Fungal and Algal Diseases

TABLE 60-2
Diagnostic Assays Currently Available for Blastomycosis in Dogs and Cats
Assay Specimen Type Target Performance
Cytologic Aspirates of affected tissues, Blastomyces dermatitidis Organisms are usually present in vari-
­examination body fluids, impression yeasts able numbers but occasionally may not
smears of skin lesions be visualized. Organisms may appear
disrupted after treatment with antifungal
drugs.
Fungal culture Aspirates or biopsies of B. dermatitidis Rarely indicated. Sensitive and specific
­affected tissues, body fluids and may be required for animals when
cytologic examination is negative. Risk
of laboratory-acquired infections. Slow
turnaround time (may require several
weeks of incubation).
Antibody serology Serum Antibodies to B. dermatitidis When present in conjunction with consis-
(gel ID) tent clinical signs, positive test results
usually indicate active infection, but
the potential for false positives exists.
Negative test results occur commonly in
dogs and cats with blastomycosis (low
sensitivity).
Antigen assay Urine, serum B. dermatitidis antigen Highly sensitive (>90%) when urine is used
(ELISA) as the test specimen. False positives are
extremely rare in dogs that lack fungal
disease, but cross-reactivity with other
fungal pathogens may occur (especially
Histoplasma), so positive results are not
diagnostic for blastomycosis.
Real-time PCR Whole blood B. dermatitidis DNA Not yet validated in adequate numbers of
assays dogs or cats with blastomycosis; useful-
ness requires further evaluation.
Histopathology Biopsy specimens from B. dermatitidis yeasts Sensitive, but yeasts may be difficult to find
­affected tissues in some infections. Special stains may
assist organism detection.

Laboratory Abnormalities
Complete Blood Count
The hemogram of dogs or cats with blastomycosis can be unre-
markable. However, a mild, normocytic, normochromic nonre-
generative anemia is present in many affected animals. Mild to
moderate neutrophilia is also present in many dogs and may be
accompanied by a mild to moderate bandemia.10,38 Mild mono-
cytosis, lymphocytosis, or lymphopenia may be detected.
cylindruria are present. Rarely, B. dermatitidis yeasts are identi-
fied in the sediment.
Cerebrospinal Fluid Analysis
CSF analysis in dogs with CNS blastomycosis can reveal
increased total nucleated cell counts and increased CSF protein
concentration.39 Nucleated cells typically consist of a mixture of
small and large mononuclear cells and neutrophils.

Serum Biochemical Tests Diagnostic Imaging

Serum biochemistry findings in animals with blastomycosis
include mild to moderate hyperglobulinemia due to a poly-
clonal gammopathy, hypoalbuminemia, and, uncommonly,
mild hypercalcemia.10,38 Hypoalbuminemia was present in 70
of 91 (77%) of dogs in one study, hyperglobulinemia in 58%,
and hypercalcemia in 14% of dogs.10
Urinalysis
The urinalysis is usually unremarkable in animals with blas-
tomycosis, but occasionally proteinuria, pyuria, hematuria, or
Plain Radiography
Radiographic patterns in dogs with blastomycosis vary con-
siderably and include unstructured, miliary, or nodular inter-
stitial patterns; a bronchointerstitial pattern; alveolar or mixed
alveolar-interstitial patterns; pulmonary mass lesions (>3 cm in
diameter); or single or multiple large (0.5 to 2.9 cm) pulmo-
nary nodules that can resemble metastatic pulmonary neopla-
sia (Figure 60-6).10,40,41 Tracheobronchial lymphadenopathy
is present in approximately 25% of dogs.40 Pleural thickening
and mild pleural effusion are uncommonly identified (≤10% of

A
B antibody responses to the BAD-1 antigen had a sensitivity of
FIGURE 60-6  Radiographic patterns in pulmonary blastomycosis. A, Lateral thoracic
radiograph from a 2-year-old female spayed Labrador retriever with a 1-week history of
cough and inappetence. A diffuse miliary nodular interstitial pattern is present. B, Lateral
thoracic radiograph from a 5-year-old female spayed German shepherd dog with leth-
argy and inappetence. An alveolar pattern is present in the caudal lung lobes, along with
marked hilar lymphadenopathy. There is also a nodular lesion in the cranial lung lobes just
ventral to the trachea. (Courtesy Daniel Cronk, University of Minnesota.)
affected dogs). Focal bronchiectasis has also been described. In
one study, only 2 of 125 dogs with blastomycosis had no ­visible
abnormalities on thoracic radiography. Findings in affected cats
include diffuse miliary or nodular interstitial patterns, lobar
consolidation, and/or pleural effusion.13,31
Radiographs of affected bone typically show osteolysis, often
accompanied by periosteal proliferation and soft tissue swelling.
Pathologic fractures may be identified (Figure 60-7). Evidence
of hypertrophic osteopathy was present bilaterally along the
humerus, radius, ulna, metacarpi, and phalanges of a dog with
a mass lesion in the right middle lung lobe.22
Advanced Imaging
Computed tomographic findings in dogs with CNS blastomy-
cosis include intra-axial, intranasal, or retrobulbar mass lesions
that are uniformly or heterogeneously contrast enhancing.25 An
intra-axial contrast-enhancing mass lesion was also described
CHAPTER 60  Blastomycosis 579
in a cat.42 Intranasal or retrobulbar mass lesions can invade
the cribriform plate or other parts of the skull, with associ-
ated osteolysis. Meningeal or periventricular contrast enhance-
ment may also be present.25,26 MRI findings have not been
extensively described. One dog had a retrobulbar mass lesion
that extended through the calvarium; the lesion was iso- to
hypointense on T2-weighted images, slightly hypointense on
T1-weighted images, and showed strong homogenous contrast
enhancement.29
Microbiologic Tests
Cytologic Examination
Cytologic examination of fine-needle aspirates, respiratory
wash specimens, or body fluids frequently reveals large numbers
of B. dermatitidis yeasts. The yeasts are 8 to 15 µm in diam-
eter, have a thick, refractile cell wall, and exhibit broad-based
budding (Figure 60-8). Daughter cells are nearly as large as the
parent cell when they detach. A pyogranulomatous inflamma-
tory response is usually present. Occasionally, organisms are
not seen; the sensitivity of transtracheal lavage for diagnosis of
pulmonary blastomycosis in two studies of 17 and 39 dogs was
76% and 69%,38,41 whereas the sensitivity of fine-needle aspira-
tion of the lung was 46 of 57 (81%).38
Serologic Diagnosis
Serologic assays that detect antibody and those that detect
B. dermatitidis antigen are available on a commercial basis in
North America. The most widely available antibody assay uses
gel immunodiffusion (ID) (see Chapter 2), which detects anti-
bodies to the B. dermatitidis A antigen. Unfortunately, gel ID
assays have variable and generally unacceptable sensitivity for
diagnosis of blastomycosis in dogs and cats, which has ranged
from 17% to 91%.10,31,38,43-45 An enzyme immunoassay that
used crude mold-phase B. dermatitidis as the antigen had a sen-
sitivity of 76.1%,43 and a radioimmunoassay designed to detect
92%.45 The specificity of the gel ID assay exceeds 95% based
on limited studies of healthy dogs or dogs with diagnoses other
than blastomycosis.44,45 However, because false-positive test
results have the potential to occur as a result of exposure and
recovery in endemic areas or as a result of cross-reactivity to
other infectious agents, diagnosis based on positive antibody
serology alone is not recommended.
An assay that detects Blastomyces cell wall galactomannan
antigen (MiraVista Diagnostics, Indianapolis, IN) has largely
replaced antibody assays for serologic diagnosis of canine blas-
tomycosis. The performance of this assay was evaluated in 46
dogs with confirmed blastomycosis.43 When urine was used, the
sensitivity was 93.5%, whereas when serum was assayed, sensi-
tivity was 87%. Only 1 of 43 control dogs without blastomyco-
sis had a positive test result. In human patients, the sensitivity of
urine galactomannan antigen testing was 90%, and specificity
was 99% in patients without fungal infections. However, 96%
of humans with histoplasmosis tested positive as a result of
cross-reactivity.46 Positive test results can also occur in dogs and
cats that have histoplasmosis, although the extent to which this
occurs requires further study. It is possible that cross-reactivity
may also occur when other mycoses are present.
Fungal Culture
B. dermatitidis can be isolated from clinical specimens on rou-
tine fungal media in the laboratory. Growth of a white mold
580 SECTION 3  Fungal and Algal Diseases
A B
FIGURE 60-7  Lateral (A) and anteropalmar (B) radiograph of the right carpus showing an osteolytic and osteoproductive (small arrow) lesion with a pathologic fracture (arrowhead)
and associated soft tissue swelling in the distal radius of a 6-year-old male neutered Labrador retriever with disseminated blastomycosis. (Image courtesy Daniel Cronk, University of
Minnesota.)
FIGURE 60-8  Cytology of a fine-needle aspirate of a skin lesion from a dog with
blastomycosis. Blastomyces yeasts (arrows) have a thick wall and exhibit broad-based bud-
ding. A pyogranulomatous inflammatory response is also present. (Image courtesy Dr. Jed
Overmann, University of Minnesota.)
typically appears after incubation for 1 to 3 weeks, but occa-
sionally incubation periods of up to 5 weeks are required. The
organism is identified based on the morphology of its conidia,
which are round to oval and attached to hyphae (see Figure
60-1). Because B. dermatitidis grows as a mycelium in the labo-
ratory, culture is a laboratory health hazard and should be per-
formed only if necessary. The laboratory should be warned of
the possibility of a dimorphic fungal infection, so that appropriate
precautions are taken.
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR assays have been developed that rapidly detect
B. dermatitidis in clinical specimens from humans,47,48 but are
not currently used routinely for diagnosis in dogs and cats. The
sensitivity of one assay was 86% when compared with culture
of clinical specimens.48 PCR assays have not been widely applied
to the diagnosis of blastomycosis in dogs and cats.
Pathologic Findings
Gross pathologic findings in dogs with blastomycosis include
thin body condition; characteristic skin lesions; bony prolifera-
tions that may be accompanied by pathologic fracture; enlarge-
ment of the peripheral and/or tracheobronchial lymph nodes;
ocular lesions; lung consolidation; and firm, pale, pulmonary
nodules or masses that range in size from 1 mm to several centi-
meters in diameter. Masses may be caseous on cut surface. Ani-
mals with CNS involvement may have masses within the brain,
secondary hydrocephalus, or retrobulbar or caudal nasal cavity
granulomas that may invade the cribriform plate and extend
along optic nerves.27,39 Rarely, pleural and/or peritoneal effu-
sion and nodular lesions within abdominal viscera have been
reported.20 Testicular and prostatic masses can also be found.27
Histopathology reveals granulomatous or pyogranulomatous
inflammatory infiltrates in a variety of organs, often with intra-
lesional budding yeasts (Figure 60-9). Multinucleated giant
cells, fibroblasts, and large numbers of lymphocytes may also
be present. Ocular lesions include uveitis, choroiditis, retinal
detachment, retinal degeneration, lens rupture, cataracts, optic
 CHAPTER 60  Blastomycosis 581

A B
FIGURE 60-9  Histopathologic findings in dogs with blastomycosis. A, Histopathology of the lymph node from a 10-year-old female spayed Australian shepherd dog with
blastomycosis. Severe pyogranulomatous lymphadenitis is present with moderate numbers of large yeast organisms. (Image courtesy Dr. Catherine Benson, University of Minnesota.)
B, Histopathology of the lung of another Australian shepherd dog that had a pulmonary and a mediastinal abscess due to Blastomyces dermatitidis. Abundant yeast organisms are
present. 1000x oil magnification. (Image courtesy Dr. Patricia Pesavento, University of California, Davis.)

neuritis, and vitritis.27,34 Secondary hepatic and renal amyloido-
sis was described in one dog.49
Organisms are not always detected in lesions, even in
untreated dogs. Special stains such as silver stains, periodic
acid–Schiff, and immunohistochemical stains can assist in the
identification of B. dermatitidis yeasts within sections,34,49,50
but failure to detect yeasts does not rule out blastomycosis.
Treatment and Prognosis
Antimicrobial Treatment
The most widely used treatment for blastomycosis in dogs
is itraconazole, which is effective as a single agent in many
dogs.38,51 Itraconazole has largely replaced the use of ketocon-
azole for treatment of blastomycosis. The recommended dose
of itraconazole for dogs is 5 mg/kg PO q24h. This dose has an
equivalent efficacy as a dose of 5 mg/kg PO q12h and a lower
rate of adverse effects such as anorexia.51 The duration of azole
treatment should be based on serial monitoring of clinical
signs and radiographic lesions (e.g., every 4 to 8 weeks). Most
dogs require at least 3 to 6 months of treatment, and some
dogs require treatment for more than 1 year, especially those
with osteoarticular infections or widespread dissemination.
Because fluconazole is less active, its use is not recommended
for treatment of human blastomycosis, unless itraconazole is
not tolerated.52 Treatment with deoxycholate amphotericin B
or lipid-complexed amphotericin B is also effective and could
be considered for dogs with severe disease with widespread
dissemination, for animals that do not respond to azole mono
therapy, or for those that do not tolerate itraconazole (see
Box 9-1).38,53 Cats with blastomycosis have also been treated
with variable success with combinations of amphotericin B
and azoles or amphotericin B alone.13,31 In human patients,
the use of deoxycholate or lipid-complexed amphotericin B is
recommended for severe pulmonary or disseminated disease,
followed by step-down itraconazole therapy after there has
been a satisfactory clinical response.52 Whether protocols that
include amphotericin B offer a survival advantage over azole
monotherapy for dogs or cats has not been well studied in a
prospective fashion. Other drugs that have been used success-
fully to treat human blastomycosis include voriconazole and
posaconazole.54,55 Because of its ability to penetrate the CNS
and eye, voriconazole has been recommended for treatment of
human CNS blastomycosis after initial treatment with lipid-
complexed amphotericin B.55
Urine Blastomyces galactomannan antigen concentra-
tion declines with effective antifungal drug treatment.43
It is not yet clear whether treatment should be continued
until antigenuria is undetectable when radiographic lesions
resolve earlier. It is possible that the immune system of some
dogs may continue to clear antigen even after treatment is
discontinued.
Supportive Care
Other treatments that may be required for treatment of dogs
or cats with severe pulmonary blastomycosis are supplemental
oxygen, nebulization and coupage, intravenous fluid therapy,
and, in some cases, mechanical ventilation.38 Mechanical ven-
tilation was used to treat 5 (4%) of 125 dogs with pulmonary
blastomycosis in one report, none of which survived.38 Dogs
with CNS involvement may require treatment with anticon-
vulsants in order to control seizure activity. The concurrent
use of systemic glucocorticoids should also be considered to
control brain inflammation and edema, although whether
this ultimately improves outcome is not known. For animals
that lack CNS involvement, NSAIDs can be used to control
pyrexia. Topical anti-inflammatory and antiglaucoma agents
may be indicated if ocular involvement is present. Enucle-
ation may ultimately be required if endophthalmitis is present
to control ocular pain and eliminate infection. Treatment of
osteomyelitis or large pulmonary granulomas that are refrac-
tory to antifungal chemotherapy may require surgical treat-
ment by amputation or lung lobectomy, respectively.
Prognosis
Cure rates of 50% to 75% have been reported in dogs with
blastomycosis.51,56 An additional 20% of dogs experience
relapse of disease after treatment is discontinued.51 Clinical
signs and radiographic lesions can worsen in the first few days
of treatment in some affected dogs,40 possibly as a result of the
inflammatory response to dying organisms. The median time
for resolution of primary radiographic patterns in dogs with
582 SECTION 3  Fungal and Algal Diseases
A B
FIGURE 60-10  Lateral thoracic radiographs from a young adult male neutered golden retriever with blastomycosis. A, A nodular soft tissue pattern is present diffusely throughout all
lung lobes, which coalesces to alveolar infiltrates in the left caudal lung lobe and right cranial lung lobe. B, Residual pulmonary infiltrates are present in the right cranial lung lobe after 14
months of itraconazole treatment. There is also a large bulla in the left caudal lung lobe (arrows).
pulmonary blastomycosis in one study was 186 days (range, 4
to >355 days).40 Significantly longer mean treatment durations
were required for radiographic improvement of large pulmo-
nary masses when compared with alveolar and interstitial pat-
terns. The most common radiographic sequela to pulmonary
blastomycosis is development of one or more pulmonary bullae.
Unstructured interstitial patterns may also persist, presumably
as a result of pulmonary fibrosis (Figure 60-10).40
Involvement of the CNS, severe lung disease, and a high band
neutrophil count are negative prognostic factors.38,51,56 In one
study, the median band neutrophil count in dogs that did not
survive was 1200 cells/µL (range, 0 to 3980 cells/µL), whereas
that in survivors was 0 cells/µL (range, 0 to 2380 cells/µL).38
In that study, 63% of 125 dogs with blastomycosis survived.
The prognosis for survival in animals with neurologic involve-
ment is particularly poor. The prognosis for resolution of endo-
phthalmitis is also poor; only 20% of eyes with endophthalmitis
respond favorably to antifungal drug treatment.33 Nevertheless,
eyes that do not undergo enucleation can ultimately progress to
phthisis bulbi after completion of treatment, without recurrence
of systemic infection.33
Immunity and Vaccination
Immunity to blastomycosis is initially dependent on phagocyte
function, especially neutrophils and alveolar macrophages,
which can clear conidia. However, once the organisms have tran-
sitioned to the yeast form, control of infection also depends on
T lymphocytes, which stimulate macrophages to kill the yeasts.52
Humoral immunity is not essential for resolution of infec-
tion. An experimental vaccine that includes a genetically engi-
neered, live-attenuated BAD-1 deletion mutant B. dermatitidis
strain protected mice from experimental infection, was safe in
25 beagles and 78 foxhounds in a field trial, and induced specific
immune responses to B. dermatitidis antigens.57,58 Although fur-
ther study is required, this vaccine holds promise for prevention
of blastomycosis in dogs that reside in hyperendemic regions of
the United States.
Prevention
Avoidance of specific foci of hyperendemicity where other dogs
or humans have contracted blastomycosis may prevent blasto-
mycosis, but this is not always possible.
Public Health Aspects
Like dogs, humans acquire B. dermatitidis infection from the
environment. Dogs are considered sentinels for human exposure
and infection,59 although whether strains that infect dogs also
infect humans requires further study. The incidence of blasto-
mycosis in dogs is approximately 8 times that in humans, and
disease has been reported in humans and dogs that reside in the
same household.59,60 Approximately 50% of human infections
are asymptomatic.52 When illness develops, it typically occurs
30 to 45 days after exposure and is often a mild, self-limiting
influenza-like illness with fever and cough. Blastomycosis is sus-
pected in endemic areas when respiratory signs persist and fail
to respond to treatment with antibacterial drugs. Radiographic
abnormalities resemble those in dogs, although hilar lymphade-
nopathy is not often seen. Chronic pulmonary blastomycosis can
resemble pulmonary neoplasia or tuberculosis. Rarely, blasto-
mycosis-associated acute respiratory distress syndrome (ARDS)
develops, which is associated with mortality rates that exceed
50%.52 Dissemination to extrapulmonary sites similar to those
involved in dogs occurs in 20% to 25% of affected humans.
Serious infections can occur in immunocompromised patients
such as those with AIDS, solid organ transplant recipients, and
humans treated with TNF-α blockers (used to treat inflamma-
tory diseases such as Crohn’s disease, psoriasis, and rheumatoid
arthritis).52,61,62
Inoculation blastomycosis that involves the skin and under-
lying bone has been described in veterinary personnel after
sharps injuries or bite wounds from dogs with blastomycosis.
One involved a needle-stick injury after a lung aspirate in a dog
with suspected blastomycosis, and another was an accident that
occurred during a necropsy.63,64 Bite wounds from dogs with

blastomycosis can result in localized disease; disseminated dis-
ease occurred in a renal transplant recipient.65-67 A veterinary
technician developed pulmonary blastomycosis after B. derma-
titidis was cultured unexpectedly in a veterinary clinic in-house
laboratory.68 Although these routes of transmission are rare,
prevention of sharps injuries, avoidance and proper management
of bite wounds, and use of accredited veterinary laboratories for
CASE EXAMPLE
Signalment: “Sam,” a 3-year-old male neutered golden
retriever from Burlingame in northern California
History: Sam was evaluated by the Veterinary Emergency and
Critical Care Service at the University of California, Davis, for
pulmonary blastomycosis, which had been diagnosed at a
local veterinary clinic. Sam had a 1-week history of lethargy
and inappetence. The day after illness was noted, Sam
was taken to a local veterinary clinic. Routine blood work
showed only anemia (hematocrit 34%), thrombocytosis
(554,000 platelets/µL), and a mildly increased serum alkaline
phosphatase activity (199 U/L). Thoracic radiographs showed
hilar lymphadenopathy, a mild diffuse miliary interstitial
pattern, and focal alveolar infiltrates in the right cranial and
left caudal lung lobe. Cytologic examination of a transtracheal
lavage specimen showed pyogranulomatous inflammation
with moderate numbers of yeasts that had morphology
consistent with Blastomyces dermatitidis. Serology (gel
immunodiffusion) for detection of antibodies to B. dermatitidis
was negative. Treatment with itraconazole (5 mg/kg PO q24h)
was commenced, and after 2 days the dose was increased to
10 mg/kg PO q24h because of lack of clinical improvement.
Subsequently the dog’s rectal temperature increased to 105°F
(40.6°C) and a day later, radiographs showed an increase in the
severity of the alveolar infiltrates. Treatment with carprofen
(2.4 mg/kg PO q12h) was initiated, and subcutaneous fluids
were administered. This was associated with resolution
of pyrexia, but the dog remained lethargic and exercise
intolerant and the owners noted right pelvic limb lameness.
The dog was referred for further evaluation.
Approximately 6 weeks before he became ill, Sam had been
taken to Georgian Bay in Ontario, Canada, for 1 week. He
had also been in the Vermont region for 2 months before
that time. Other travel had been limited to various bayside
and mountainous parts of northern California. The owner’s
brother’s dog, which had traveled with Sam to Ontario and
Vermont, had also been diagnosed with blastomycosis 3
weeks earlier.
Physical Examination:
Body Weight: 41.5 kg.
General: Quiet, alert and responsive. T = 103.5°F (39.7°C), HR =
120 beats/min, panting, CRT <2 s, mucous membranes were
moist and slightly cyanotic.
Eyes, Ears, Nose, and Throat: No clinically significant
abnormalities were detected. A fundoscopic examination
with dilated pupils was unremarkable.
Musculoskeletal: BCS 4/9; no obvious lameness or pain on
palpation was appreciated.
CHAPTER 60  Blastomycosis 583
isolation of microorganisms from dogs and cats may reduce the
risk of inoculation or laboratory-acquired blastomycosis in vet-
erinary staff. The bandaging of skin lesions has been discouraged
because it has the potential to promote transition of the organism
to the mold form, but this has not been documented to occur.
The body of deceased pets with blastomycosis should be disposed
of promptly and by cremation.
Respiratory: Increased respiratory effort was present. Lung
sounds were increased diffusely on thoracic auscultation
and partially obscured the heart sounds. Markedly
increased respiratory effort was evident when the dog’s
gait was assessed as part of an orthopedic examination. An
intermittent nonproductive cough was also appreciated.
All Other Systems: No clinically significant abnormalities were
detected. No obvious neurologic deficits were noted, but a full
neurologic examination was not performed.
Laboratory Findings: Blood oxygen saturation (pulse
oximetry) = 91%, improved to 95% with flow-by
supplemental oxygen
CBC:
HCT 37.3% (40%-55%)
MCV 71.2 fL (65-75 fL)
MCHC 35.1 g/dL (33-36 g/dL)
WBC 14,640 cells/µL (6000-13,000 cells/µL)
Neutrophils 11,273 cells/µL (3000-10,500 cells/µL)
Band neutrophils 586 cells/µL
Lymphocytes 1318 cells/µL (1000-4000 cells/µL)
Monocytes 1318 cells/µL (150-1200 cells/µL)
Eosinophils 146 cells/µL (0-1500 cells/µL)
Platelets 387,000 platelets/µL (150,000-400,000 platelets/µL)
Rare slight toxicity of band neutrophils was reported.
Serum Chemistry Profile:
Sodium 148 mmol/L (145-154 mmol/L)
Potassium 5.0 mmol/L (4.1-5.3 mmol/L)
Chloride 116 mmol/L (105-116 mmol/L)
Bicarbonate 15 mmol/L (16-26 mmol/L)
Phosphorus 6.6 mg/dL (3.0-6.2 mg/dL)
Calcium 9.9 mg/dL (9.9-11.4 mg/dL)
BUN 11 mg/dL (8-21 mg/dL)
Creatinine 0.5 mg/dL (0.5-1.6 mg/dL)
Glucose 81 mg/dL (60-104 mg/dL)
Total protein 5.6 g/dL (5.4-7.4 g/dL)
Albumin 1.8 g/dL (2.9-4.2 g/dL)
Globulin 3.8 g/dL (2.3-4.4 g/dL)
ALT 15 U/L (19-67 U/L)
AST 32 U/L (21-54 U/L)
ALP 148 U/L (15-127 U/L)
GGT 0 U/L (0-6 U/L)
Cholesterol 318 mg/dL (135-345 mg/dL)
Total bilirubin 0.4 mg/dL (0-0.4 mg/dL).
Urinalysis: SGr 1.015; pH 5.0, no protein (SSA), 1+ bilirubin,
no hemoprotein, no glucose, no WBC, rare RBC/HPF, rare
transitional epithelial cells, and a few amorphous crystals
were seen.
Continued
584 SECTION 3  Fungal and Algal Diseases
Imaging Findings:
Thoracic Radiographs: A nodular soft tissue pattern was
present diffusely throughout all lung lobes, which coalesced
to alveolar infiltrates in the left caudal lung lobe and right
cranial lung lobe (see Figure 60-10). The cardiac silhouette
was mildly enlarged. Increased soft tissue opacity was noted
in the perihilar region.
Abdominal Ultrasound: The spleen was mildly enlarged, and
there was mild mesenteric lymphadenopathy.
Diagnosis: Acute, severe pulmonary blastomycosis.
Treatment: Sam was placed in an oxygen cage with an inspired
oxygen concentration (FiO2) of 40% to 60%. An arterial blood
gas revealed a pH of 7.301, pCO2 of 36.6 mm Hg, pO2 of
79.1 mm Hg, HCO3 of 16.8 mmol/L, base deficit of 7.8 mmol/L,
and measured O2 saturation of 92.8%. The dog was also
treated with crystalloid fluids (lactated Ringer’s solution with
20 mEq/L KCl; 80 mL/hr, IV) and nebulization and coupage
(q6h). For the first 24 hours, respiratory rate ranged from 90
to 105 breaths/min and a nonproductive cough occurred
every few hours. The dog drank water, but was inappetent.
Treatment with lipid-complexed amphotericin B was
initiated (1 mg/kg IV on a Monday-Wednesday-Friday basis)
and carprofen administration continued. By day 4, Sam was
afebrile, eating, and oxygen saturation was 96% with an FiO2
of 46%. However, increased respiratory effort developed
when oxygen supplementation was discontinued, and
arterial blood gas analysis showed a pH of 7.335, pCO2 of 27.2
mm Hg, pO2 of 65.8 mm Hg, HCO3 of 13.9 mmol/L, and base
deficit of 10.6 mmol/L (room air). By day 5, Sam was playing
with toys in the oxygen cage, and his appetite was excellent.
Treatment with itraconazole (5 mg/kg PO q24h) was resumed
in addition to the amphotericin B. By day 8, oxygen saturation
was 95% with an FiO2 of 30%. Thoracic radiographs showed
persistent radiographic abnormalities, organization of the
alveolar infiltrates, and apparent bronchiectasis. Survey
radiographs of the appendicular skeleton showed no
evidence of osteomyelitis. The dog was discharged after 11
days of hospitalization (cumulative amphotericin B dose of
6 mg/kg). An additional seven treatments of amphotericin
B were then administered at the local veterinary clinic. Serial
renal panels showed no evidence of nephrotoxicity.
SUGGESTED READINGS
Arceneaux KA, Taboada J, Hosgood G. Blastomycosis in dogs: 115
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Crews LJ, Feeney DA, Jessen CR, et al. Radiographic findings in dogs
with pulmonary blastomycosis: 125 cases (1989-2006). J Am Vet
Med Assoc. 2008;232:215-221.
Smith JA, Kauffman CA. Blastomycosis. Proc Am Thorac Soc. 2010;
7:173-180.
Spector D, Legendre AM, Wheat J, et  al. Antigen and antibody test-
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At a recheck 1 month after the date of first evaluation, Sam re-
mained somewhat lethargic and exercise intolerant, but had
gained 3 kg. The frequency of cough had decreased from
approximately 8 times a day to once or twice daily over the
previous 2 weeks. An arterial blood gas showed a pO2 of 93.5
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31. Miller PE, Miller LM, Schoster JV. Feline blastomycosis: a report
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32. Bromel C, Sykes JE. Epidemiology, diagnosis, and treatment
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33. Bloom JD, Hamor RE, Gerding Jr PA. Ocular blastomycosis
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39. Nafe LA, Turk JR, Carter JD. Central nervous system involvement of
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42. Smith JR, Legendre AM, Thomas WB, et  al. Cerebral Blas-
tomyces dermatitidis infection in a cat. J Am Vet Med Assoc.
2007;231:1210-1214.
43. Spector D, Legendre AM, Wheat J, et al. Antigen and antibody test-
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44. Legendre AM, Becker PU. Evaluation of the agar-gel immunodif-
fusion test in the diagnosis of canine blastomycosis. Am J Vet Res.
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45. Klein BS, Squires RA, Lloyd JK, et  al. Canine antibody response
to Blastomyces dermatitidis WI-1 antigen. Am J Vet Res.
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48. Babady NE, Buckwalter SP, Hall L, et al. Detection of Blastomy-
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57. Wuthrich M, Krajaejun T, Shearn-Bochsler V, et al. Safety, toler-
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58. Wuthrich M, Filutowicz HI, Klein BS. Mutation of the WI-1 gene
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exposure near one’s domicile. WMJ. 2001;100:43-45.
61. Pappas PG. Blastomycosis in the immunocompromised patient.
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62. Gauthier GM, Safdar N, Klein BS, et  al. Blastomycosis in solid
organ transplant recipients. Transpl Infect Dis. 2007;9:310-317.
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1994;205:968.
64. Graham Jr WR, Callaway JL. Primary inoculation blastomycosis in
a veterinarian. J Am Acad Dermatol. 1982;7:785-786.
65. Gnann Jr JW, Bressler GS, Bodet 3rd CA, et al. Human blastomy-
cosis after a dog bite. Ann Intern Med. 1983;98:48-49.
66. Butka BJ, Bennett SR, Johnson AC. Disseminated inoculation
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67. Jaspers RH. Letter: Transmission of Blastomyces from animals to
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68. Cote E, Barr SC, Allen C. Possible transmission of Blastomy-
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1997;210:479-480.
CHAPTER 61
Histoplasmosis
Jane E. Sykes and Joseph Taboada
Overview of Histoplasmosis
First Described: 1905, Panama (by Samuel Darling), in a per-
son from Martinique, where it was initially mistaken for a
protozoal pathogen.1
Cause: Histoplasma capsulatum (an ascomycete)
Affected Hosts: Many mammalian species including dogs,
cats, and humans
Geographic Distribution: Worldwide; but especially the Ohio,
Missouri, Tennessee, and Mississippi river valleys of the
United States; and Latin America
Mode of Transmission: Inhalation of microconidia from the
environment
Major Clinical Signs: Cough, tachypnea, organomegaly, gas-
trointestinal signs, pallor; signs are often non-specific in
cats.
Differential Diagnoses: Other deep mycoses, protothecosis,
mycobacterial infections, hemic neoplasia, leishmaniosis,
histiocytic colitis
Human Health Significance: H. capsulatum also causes dis-
ease in humans but direct transmission from animals to
humans does not occur
Etiology and Epidemiology
Histoplasma capsulatum is a dimorphic, soil-borne fungus that
is found worldwide, but especially along the Mississippi, Mis-
souri, Tennessee, and Ohio river valleys of the United States, as
well as in Latin America (Figure 61-1). Endemic areas within
the United States span the central portion of the United States
from western Virginia to central Texas. Disease has also been
reported in cats or dogs from northern California2; Brazil3;
Italy4; Queensland in Australia5; Japan6; and western Can-
ada.7,8 Eight clades of the organism have been identified by
genetic analysis: North American (class 1 and class 2), Latin
American (group A and group B), and one each of Australian,
Indonesian, Eurasian, and African. These may vary in virulence.
All except the Eurasian clade, which is derived from the Latin
American group A clade, may be distinct phylogenetic species.9
A possible ninth clade, which is related to, but distinct from,
North American class 1 also appears to infect cats in North Amer-
ica outside the accepted endemic range.10 Three variants have
also been described based on pathogenicity and morphology—
H. capsulatum var. capsulatum, H. capsulatum var. duboisii,
and H. capsulatum var. farciminosum—although the phyloge-
netic value of these variants has been debated.6,9 H. capsulatum
var. duboisii occurs primarily in Africa and Japan. H. capsula-
tum var. farciminosum infections occur primarily in horses, but
have also been reported in humans and dogs.
H. capsulatum grows best in soil where temperatures are
between 22°C and 29°C, where annual rainfall is 35 to 50 inches,
and where relative humidity is 67% to 87%. These conditions
are typically found between latitudes of 45 degrees north to
30 degrees south.11 H. capsulatum can be found in the intestinal
tracts and guano of bats, which are the primary reservoir of the
organism and serve to disseminate it geographically.12 Bat caves
can maintain perfect growth conditions for H. capsulatum.
Although H. capsulatum can be found in high concentrations in
decaying avian guano (especially around blackbird or starling
roosts and chicken coops), it is not found in fresh feces or shed
in the feces of birds.
Cats are as susceptible, or slightly more susceptible, to histo-
plasmosis than dogs. Affected cats can be as young as 2 months
and, in some cases, older than 15 years.13 The mean age of
affected cats has varied from around 4 years to 9 years.14 For
dogs, a mean age of 4.3 years was reported (range, 5 months to
10 years).15 Dogs in the 2- to 4-year and 4- to 7-year age groups
were more likely to develop histoplasmosis than dogs less than
2 years of age.16 Sporting or working dogs may be at greater
risk of infection because of increased exposure; in a study using
the Veterinary Medical Database, pointers were strongly over-
represented.16 Weimaraners and Brittany spaniels were also at
increased risk. Among cats, Persian cats may be slightly over-
represented. A sex predisposition has not been clearly iden-
tified in dogs or cats, but more female than male dogs were
affected in some case series.14,15,17-19 This contrasts to human
histoplasmosis and other systemic mycoses in dogs, which affect
males more often than females.20 The overwhelming majority of
cats with histoplasmosis are retrovirus negative, but a signifi-
cant percentage of cats in two studies (16% and 28%, respec-
tively) were co-infected with FeLV.13,14 A small percentage of
cats have underlying comorbidities such as lymphoma, feline
infectious peritonitis, or a history of glucocorticoid treatment.13
Comorbidities described in dogs include dirofilariasis15 and
metastatic carcinoma.6 Disease can occur in cats that are housed
exclusively indoors.
Clinical Features
Signs and Their Pathogenesis
In the soil, the mycelial phase of H. capsulatum forms macro-
conidia and microconidia (Figure 61-2). The microconidia are
thought to be the form inhaled by mammalian hosts, because
587
588 SECTION 3  Fungal and Algal Diseases

they are small enough (2 to 5 µm) to enter the terminal bronchi-
oles and alveoli. Within the lungs (i.e., at a temperature of 37°C),
the microconidia transition to a unicellular yeast, which repli-
cates by budding. The fungus binds to CD11-CD18 integrins
Highly endemic
Moderately endemic
Suspected endemic
FIGURE 61-1  Approximate geographic distribution of histoplasmosis in the
Americas.
on alveolar macrophages and is phagocytized. It then replicates
within these cells through control of the phagolysosomal envi-
ronment and ultimately destroys them, with subsequent replica-
tion in other resident alveolar macrophages and inflammatory
phagocytes that are recruited to the lung.21 Acquisition of iron
by the fungus is essential for growth in  vivo.22 Some animals
control the initial infection but remain latently infected with
small numbers of yeasts. Subsequent immune suppression can
lead to reactivation of infection years later. Thus, the incubation
period can range from 2 to 3 weeks to as long as several years.
The clinical manifestations and rate of disease progression
depend on the host immune response to infection, the infectious
dose, and the Histoplasma strain, which can vary geographi-
cally. The vast majority of infections are probably subclinical.23
Replication of the organism leads to a granulomatous inflam-
matory response in the lungs. Occasionally, the inflammatory
response is followed by fibrosis and scarring. The yeasts migrate
to local lymph nodes (such as the hilar lymph node) and other
tissues that contain mononuclear cells, such as the liver and
spleen. When the cell-mediated immune response is defective
or absent, disseminated disease occurs (referred to in human
patients as progressive disseminated histoplasmosis [PDH]20),
which may be acute or chronic. In addition to lymph nodes,
liver, and spleen, other common sites of dissemination are the
bone marrow, small and/or large intestinal tract, pancreas, skin,
bones, central nervous system (CNS), and eyes. Dogs with histo-
plasmosis in the United States appear to be particularly suscepti-
ble to intestinal involvement. Severe infiltration of the small and
large intestines leads to malabsorption, diarrhea, severe weight
loss, and hematochezia, often without evidence of respiratory
involvement.
Clinical signs in cats are commonly vague and nonspecific,
such as weight loss, inappetence, weakness, dehydration, and
fever.13,14 Approximately 40% of cats show respiratory signs
such as dyspnea and tachypnea, and to a lesser extent, cough
and nasal discharge. However, respiratory signs may be absent

Lung

Lymph node Blood

FIGURE 61-2  Life cycle of Histoplasma capsulatum. In the environment, the organism forms macroconidia (arrowhead, inset) and microconidia (arrows, inset). The microconidia are
thought to be inhaled into the lungs of mammalian hosts. Within the lung, the organisms transform into yeasts that infect alveolar macrophages and then disseminate to local lymph nodes
and the systemic circulation.
 CHAPTER 61  Histoplasmosis 589

in some cats with dissemination of infection to nonpulmonary masses, and/or pain on palpation of the abdomen. Ocular signs
sites. Ocular signs such as chorioretinitis and/or posterior or include chorioretinitis, optic neuritis, anterior uveitis, retinal
anterior uveitis occur in approximately one quarter of cats with detachment, panophthalmitis, and glaucoma (Figure 61-4).
histoplasmosis, and around 20% of cats have signs of skeletal In dogs, lethargy, fever, thin body condition, and pale mucous
involvement.13,14,17 Nodular or ulcerated and draining skin membranes are most often found on physical examination. Other
lesions, peripheral lymphadenopathy, vomiting, diarrhea, oral physical examination abnormalities include dehydration, hepa-
ulceration, myelopathy, and/or hematuria due to bladder wall tomegaly, icterus, and/or ascites; tachypnea, increased respira-
involvement have also been reported.4,14,17,24-29 Clinical signs tory effort, and increased lung sounds; lymphadenomegaly that
associated with gastrointestinal involvement are much less likely involves one or more peripheral lymph nodes; abnormal stool
to occur in cats than in dogs. A high proportion of affected cats quality on rectal examination (diarrhea, hematochezia, or melena);
that were necropsied had severe disseminated disease.13 nasal and ocular discharge; tachycardia; tongue lesions; and
In dogs, diarrhea, decreased appetite, weight loss, lethargy, cutaneous nodules, ulceration, or draining tracts.5,8,15,18,30,37,38
fever (up to 40°C or 104°F), and mucosal pallor are the most Ocular examination can reveal uveitis, evidence of optic neuri-
common clinical signs.7,8,15,18 In addition to inappetence, weight tis, or chorioretinitis with retinal detachment.18,39,40 One dog
loss, and diarrhea, other clinical signs referable to the gastro- was seen for lameness, hyperemia, and edema of the left pelvic
intestinal tract include melena, tenesmus, hematochezia, dys- limb because of a sclerosing pyogranulomatous inflammatory
chezia, and/or increased frequency of defecation.8,15,18 Profuse mass in the inguinal region that resulted in venous occlusion.37
diarrhea may be chronic and persist for several months. Hilar Rarely, neurologic signs such as ataxia, obtundation, head tilt,
lymphadenopathy is common, and may be an incidental find-
ing on thoracic radiographs, or it may be severe and contrib-
ute to signs of cough. Other signs include respiratory difficulty,
icterus, vomiting, hepatomegaly, lymphadenomegaly, nasal dis-
charge, ocular signs, polyuria and polydipsia, lameness due to
osteomyelitis, and neurologic signs such as seizures or paralysis/
paresis.8,15,30-32 Affected dogs in Japan have had chronic cutane-
ous or gingival lesions in the absence of pulmonary or gastro-
intestinal involvement,33-36 although disseminated disease has
also been described.6 Skin nodules were also reported in an Aus-
tralian dog with histoplasmosis.5 Varied clinical manifestations
of histoplasmosis in different parts of the world as a result of
strain variation in H. capsulatum also occur in humans.20

Physical Examination Findings

Physical examination findings in cats with histoplasmosis can
be minimal or can include thin body condition or emaciation;
fever; pale and/or rarely icteric mucous membranes; peripheral
lymphadenopathy; respiratory signs such as dyspnea, tachy-
pnea, and increased or decreased lung sounds; single or multiple A
cutaneous nodules, ulcerations, and/or draining tracts (Figure
61-3); lameness; and detection of organomegaly, abdominal

FIGURE 61-3  Cutaneous lesion on the eyelid of a cat with histoplasmosis. (Courtesy
Dr. Amy Grooters, Louisiana State University.)
B
FIGURE 61-4  Ocular changes in cats with histoplasmosis. A, Blepharitis. B, Granulo-
matous chorioretinitis with multifocal pigmented lesions. (A, From Ketring KL, Glaze MB.
Atlas of Feline Ophthalmology. 2nd ed. Hoboken, NJ: Wiley-Blackwell; 2012. B, Courtesy of
Dr. Mary Belle Glaze, Gulf Coast Animal Eye Clinic, Houston, TX.)
590 SECTION 3  Fungal and Algal Diseases

nystagmus, strabismus, seizures, facial paralysis, and tetraparesis
may be present on physical examination as a result of hepatic
encephalopathy or meningoencephalitis.40-42
Diagnosis
Laboratory Abnormalities
Complete Blood Count
The CBC in dogs and cats with histoplasmosis reflects the pres-
ence of systemic inflammation, gastrointestinal hemorrhage,
and/or bone marrow infiltration by fungal organisms. In both
dogs and cats, anemia is a common abnormality, may be mild
to severe, and is usually normocytic, normochromic, and non-
regenerative.14,15,17,18,43,44 Increased numbers of nucleated
erythrocytes occur in some affected dogs.8,44 The total white
cell count or neutrophil count may be normal, increased, or
decreased.15,17,18,43 Most affected cats have a normal or decreased
white cell count,14,43 but leukocytosis due to neutrophilia
and monocytosis may be more common in affected dogs.15,44
Increased numbers of band neutrophils with toxic neutrophils,
lymphopenia, monocytopenia, and thrombocytopenia can be
present in some severely affected animals.14,17,18,43,45 Thrombo-
cytopenia can be a result of platelet consumption, sequestration,
and/or myelophthisis. Rarely, eosinophilia is present.46
Serum Biochemical Tests
Mild to severe hypoalbuminemia is present in most (>75%)
affected dogs and cats. Cats with liver involvement may have
increased activities of serum ALT and AST.17,43 Hyperbilirubi-
nemia and increased serum ALP activity are rarely reported.17
Hypercalcemia and hyperglobulinemia have been described in
a few affected cats.17 Hyperglobulinemia; mild to moderate
increases in the activity of serum ALT, AST, ALP, and GGT;
and hyperbilirubinemia can be found in dogs.

B
FIGURE 61-5  Radiographic abnormalities in feline pulmonary histoplasmosis. A, Lateral thoracic radiograph from an 8-month-old intact male domestic shorthair cat with dis-
seminated histoplasmosis. There is a diffuse bronchointerstitial pulmonary pattern and evidence of sternal lymphadenopathy (arrow). In the viewable abdomen, marked splenomegaly
can be appreciated. B, Lateral thoracic radiograph from a 7-month-old male neutered domestic shorthair cat with histoplasmosis that was evaluated for tachypnea and dyspnea of
10 days duration. There is a diffuse, severe, interstitial-to-coalescing and patchy pulmonary pattern that obscures visualization of the pulmonary vasculature and cardiac contours. (Courtesy
of Lorrie Gaschen, Louisiana State University.)

Urinalysis
Results of urinalysis are usually within normal limits, but low
urine specific gravity and bilirubinuria may be present in dogs
with hepatic involvement.
Coagulation Testing
A few dogs with acute PDH have had coagulation abnormalities
characterized by increased PT and PTT and fibrin degradation
product or D-dimer concentrations.31,44 Coagulation abnormal-
ities in dogs with histoplasmosis may result from disseminated
intravascular coagulation and possibly severe hepatic involve-
ment (see also Canine Case Example).
Diagnostic Imaging
Plain Radiography
In cats, thoracic radiographic findings in histoplasmosis usually
consist of diffuse, linear, nodular, or miliary interstitial pat-
terns, but mixed interstitial-alveolar-bronchial patterns, pleu-
ral effusion, or an absence of abnormal findings can also occur
(Figure 61-5).14,19,43,47 Plain thoracic radiographs in dogs may
show alveolar, interstitial, and/or bronchial patterns, tracheo-
bronchial lymphadenopathy, lung lobe consolidation, and/or
rarely pleural effusion.31,45,47,48 Lesions in dogs sometimes min-
eralize, which does not seem to occur in cats. Abdominal radio-
graphs may show hepatomegaly, splenomegaly, or decreased
serosal detail due to ascites. Radiographic evidence of osteomy-
elitis may be present in cats, with associated soft tissue swell-
ing and sometimes pathologic fractures. Osteomyelitis is rarely
described in dogs.30,32
Sonographic Findings
Findings on abdominal sonographic examination of dogs with
histoplasmosis include hepatic enlargement and hypoecho-
genicity, abdominal lymphadenopathy, splenomegaly, and asci-
tes. A thickened intestinal wall may be present with disruption
of bowel wall architecture. Adrenomegaly, abdominal lymph-
adenopathy, and occasionally renomegaly have been described
in cats.14
Endoscopy
Colonoscopic findings in dogs with colonic histoplasmosis
include irregularity, ulceration, and increased granularity and
friability of the colonic wall (Figure 61-6).
Microbiologic Tests
Diagnostic assays currently available for histoplasmosis in dogs
and cats are described in Table 61-1.
Cytologic Examination
Cytologic examination of aspirates or impression smears of
affected tissues (e.g., fine-needle aspirates of liver, spleen,
lymph nodes, lung, bone marrow), rectal scrapings, or Cytospin
preparations of lung wash specimens or body fluids (e.g., CSF,
synovial fluid, ascites, or pleural effusion) usually reveals pyo-
granulomatous or granulomatous inflammation. H. capsulatum
yeasts can be seen extracellularly and intracellularly (usually
within mononuclear phagocytes). Most extracellular organ-
isms are probably an artifact of smear preparation. Occasion-
ally one or more organisms are seen in circulating monocytes,
neutrophils, or eosinophils in severely ill animals with dissemi-
nated disease (Figure 61-7). Cytologic evidence of fungemia was
detected in nearly 20% of affected cats in one study.13 The bone
CHAPTER 61  Histoplasmosis 591
marrow is a common site to yield organisms in cats, even when
hematologic abnormalities are minimal.
Cytologically, the yeasts are 2 to 4 µm in diameter, oval,
have a basophilic center, and are surrounded by a clear halo,
which results from shrinkage artifact (see Figure 61-7). They
are similar in size to Sporothrix spp., although different in
shape. A variety of stains can be used, such as Diff-Quik and
Wright stains. The organism is not encapsulated, despite its
name. Organisms may not be identified within chronic, fibros-
ing lesions.37,49
Serologic Testing
Both antibody and antigen (ELISA) assays are available for
serodiagnosis of histoplasmosis. Antibody assays for Histo-
plasma capsulatum are based on gel immunodiffusion or com-
plement fixation and have had poor diagnostic sensitivity.13,15,17
False positives reportedly occur in recovered animals, although
the specificity of antibody assays in dogs and cats is not well
documented. In human patients, acute and convalescent anti-
body testing is useful for diagnosis of acute pulmonary histo-
plasmosis, and a single positive antibody titer is used as an aid
for diagnosis of chronic histoplasmosis. In particular, a posi-
tive CSF antibody titer can be useful for diagnosis of culture-
negative chronic H. capsulatum meningitis. An ELISA assay for
H. capsulatum antigen (MiraVista Diagnostics, Indianapolis,
IN) is widely used in human patients for diagnosis of histoplas-
mosis, and urine is the preferred specimen for testing because it
results in a higher sensitivity than when serum is assayed.49 In
human patients with AIDS and severe PDH, the sensitivity of
the assay is 95% when urine is used, and 86% when serum is
used.49 The sensitivity is lower in human patients with pulmo-
nary histoplasmosis, but use of a combination of urine antigen
and serum antigen increases sensitivity to 83%.50 False positives
occur in people with other disseminated mycoses, including
blastomycosis, penicilliosis, and coccidioidomycosis,51,52 so the
FIGURE 61-6  Colonoscopy image from a dog with disseminated histoplasmosis with
large intestinal involvement. The mucosa is thickened and granular. (Courtesy Dr. Michael
Willard, Texas A&M University.)
592 SECTION 3  Fungal and Algal Diseases

TABLE 61-1
Diagnostic Assays Currently Available for Histoplasmosis in Dogs and Cats
Assay Specimen Type Target Performance
Cytologic Aspirates of affected Histoplasma capsulatum Organisms are usually present but, in the absence of
examination tissues, bone marrow as- organisms (usually in experience, may be confused with protozoa such
pirates, sometimes blood large numbers) within as Leishmania or other fungi such as Sporothrix
smears or buffy coat mononuclear phago- schenckii. False negatives can occur in animals with
preparations, body fluids cytes and granulocytes very chronic clinical manifestations.
Fungal culture Aspirates or biopsies of af- H. capsulatum Rarely indicated. Sensitive and specific and may be
fected tissues, body fluids, required for animals with chronic histoplasmosis
whole blood when cytologic examination is negative. Risk of
laboratory-acquired infections. Slow turnaround
time (several weeks of incubation may be required).
Antibody serol- Serum Antibodies to Positive test results indicate exposure but do not
ogy (complement H. capsulatum indicate active infection or correlate with clinical
fixation or gel im- disease. Negative test results occur commonly in
munodiffusion) animals with histoplasmosis (low sensitivity).
Antigen assay Urine, serum H. capsulatum antigen Sensitivity and specificity require further evaluation
(ELISA) in dogs and cats, although the assay appears to be
sensitive and specific in cats when urine is tested.
Cross-reactivity with other fungal pathogens may
occur, so positive results are not diagnostic for his-
toplasmosis. Cross-reactivity is seen with blastomy-
cosis, a systemic fungal infection with an overlap-
ping geographic range.
Real-time PCR See fungal culture H. capsulatum DNA Not yet validated in adequate numbers of dogs or
assays cats with histoplasmosis; usefulness requires further
evaluation.
Histopathology Biopsy specimens from H. capsulatum yeasts Yeasts can be difficult to find in chronic infections.
affected tissues Special stains may assist organism detection.

assay must always be used in conjunction with other diagnostic
assays in order to determine the identity of the infecting fungal
pathogen. Antigen levels become undetectable with successful
treatment.53 The antigen assay has also been used for the diag-
nosis of histoplasmosis in cats and dogs.37,54 In one study, the
urine of 17 of 18 cats with confirmed histoplasmosis was posi-
tive, whereas none of 26 cats with other diagnoses were posi-
tive.54 Additional studies that evaluate larger numbers of cats
and dogs with and without confirmed histoplasmosis, as well
as those with other fungal infections, are required to further
establish the sensitivity and specificity of this assay.
Fungal Culture
H. capsulatum can be isolated from clinical specimens on rou-
tine fungal media. There is a risk of laboratory-acquired infec-
tion when the organism grows in the mycelial form on artificial
media, so culture should be performed only if necessary, and
the laboratory should be warned of the possibility of a dimor-
phic fungal infection, so that appropriate precautions are taken.
Although most cultures are positive within 2 or 3 weeks, growth
may require up to 6 weeks of incubation. The organism is iden-
tified based on morphology (see Figure 61-2).
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR assays have been developed for rapid detec-
tion of H. capsulatum in clinical specimens from affected
humans55,56 but currently are not used routinely for diagnosis.
The sensitivity of one assay was 73% when compared with cul-
ture of clinical specimens.55 PCR assays have not been widely
applied to the diagnosis of histoplasmosis in dogs and cats; fur-
ther studies are required to determine their usefulness.
Pathologic Findings
Gross pathologic findings in dogs and cats with histoplasmosis
consist of miliary nodules or larger focal lesions in the lungs and
other organs, and enlarged lymph nodes, which may be mineral-
ized in dogs with chronic disease. In dogs, the intestinal tract may
be thickened and focal or diffuse mucosal hemorrhage may be
present. The liver and spleen may be enlarged with rounded bor-
ders, and the liver may be mottled with a reticulated appearance.
Pleural and peritoneal effusions are occasionally described.45
Histopathology yields pyogranulomatous or granulomatous
inflammation with intralesional yeasts in a variety of organs
(Figure 61-8). Lymphocytes and plasma cells may be present,
and in some animals with chronic histoplasmosis, histologic
evidence of fibrosis or (in dogs) mineralization is apparent.37,45
Special stains such as Gomori’s methenamine silver or periodic
acid–Schiff can aid detection of the yeasts in tissues.
Treatment and Prognosis
Antimicrobial Treatment
Antifungal drugs with activity against H. capsulatum include the
azole antifungal drugs ketoconazole, itraconazole, and fluconazole

A Deoxycholate or lipid-complexed amphotericin B should
B decisions.
FIGURE 61-7  A, Blood smear from a dog with Histoplasma capsulatum fungemia.
Three organisms can be seen in a circulating neutrophil (arrow). Wright’s stain, 1000×
magnification. B, Fine-needle aspirate cytology from the right popliteal lymph node of
the cat in Figure 61-5, A. There is pyogranulomatous inflammation with large numbers
of intracellular yeast organisms with morphology consistent with that of H. capsulatum.
Wright’s stain, 1000× magnification. (Courtesy Angela Royal, Louisiana State University.)
(see Chapter 9 for more information and dosing for antifungal
drugs). Itraconazole and fluconazole are preferred to ketoconazole
since they are more effective and less likely to cause toxicity.17 In
human patients, itraconazole has consistently been more effective
than fluconazole. An increased efficacy of itraconazole when com-
pared with fluconazole is likely in dogs and cats as well, although
in the authors’ experiences, some animals may be cured with flu-
conazole monotherapy. Limited data suggest that posaconazole
may be an effective salvage treatment in human patients with
histoplasmosis that fail to respond to treatment with other anti-
fungal drugs, including fluconazole, itraconazole, amphotericin B,
and voriconazole.57 Development of resistance to fluconazole and
voriconazole has been documented during treatment of human
patients with fluconazole, but not with posaconazole.58
CHAPTER 61  Histoplasmosis 593
FIGURE 61-8  Histopathology of the liver of a 5-month-old intact male redbone
coonhound with disseminated histoplasmosis. There is severe granulomatous inflamma-
tion with massive numbers of intrahistiocytic yeast organisms.
be used initially to treat dogs or cats with severe acute pulmo-
nary, acute disseminated, or CNS disease, after which treatment
should be continued with itraconazole. If funds or patient fac-
tors do not permit itraconazole treatment, fluconazole can be
used as a second-line agent.
The length of treatment and prognosis depend on the
severity and chronicity of disease and the immune status of
the host. At least 6 months of treatment is generally required.
Many animals require treatment for 12 months or longer, and
those with chronic disease may require treatment for more
than 2 years. The decision to discontinue treatment should
be based on lesion resolution (which may require serial imag-
ing studies). Resolution of positive urine antigen titers might
also be used to decide when to discontinue treatment, but fur-
ther study is required to determine whether serial monitoring
of H. capsulatum antigenuria is useful in making treatment
Supportive Treatment
Other supportive treatments that may be required depend on
the extent of infection and include intravenous crystalloid
fluids, supplemental oxygen, blood transfusions for severely
anemic animals, nutritional support, antiemetics, and medi-
cations to manage consequences of hepatic failure such as
hepatic encephalopathy. Topical glucocorticoids and atropine
may be required for animals with uveitis, and enucleation may
be required to manage persistent ocular infection and pain.
The role of systemic glucocorticoids for treatment of chronic
manifestations of histoplasmosis is controversial, and they
are not recommended for treatment of active or disseminated
histoplasmosis.
Immunity and Vaccination
Immunity to H. capsulatum is dependent on a Th1 immune
response. Production of IFN-γ activates macrophages to destroy
the fungus.59 There is currently no vaccine for prevention of
histoplasmosis, but there is interest in development of a vaccine
for at-risk human patients because of increasing incidence of
the disease.
594 SECTION 3  Fungal and Algal Diseases
Prevention
Prevention of histoplasmosis involves avoidance of bat caves
and areas previously inhabited by avian species in endemic
areas. Nevertheless, histoplasmosis has occurred in animals
housed exclusively indoors, and widespread exposure and
recovery likely occurs in endemic areas.
Public Health Aspects
As many as 80% of young adults in endemic areas have been pre-
viously infected by H. capsulatum, but only 1% develop symp-
toms.49 Risk factors identified for human histoplasmosis include
exposure to soil disrupted as a result of excavation, exploration
of caves inhabited by bats, or renovation of buildings inhabited
by birds or bats. Males are predisposed in a 4:1 ratio.20 Several
forms of the disease have been described, which include acute
pulmonary histoplasmosis, chronic pulmonary histoplasmosis
(which may be cavitary or noncavitary), and acute or chronic
CANINE CASE EXAMPLE
Signalment: “Murphy,” a 5-month-old intact male redbone
coonhound from northern California.
History: Murphy was evaluated at the University of California,
Davis, Veterinary Medical Teaching Hospital for a 1-week
history of increased respiratory effort, hypersalivation, a
single episode of vomiting, and lethargy. After the first day
of illness he was taken to a local veterinary clinic where fever
(103.2°F or 39.6°C), pale mucous membranes, and a cranial
abdominal mass were noted on physical examination.
Abdominal radiographs revealed moderate hepatomegaly
and mild splenomegaly. He was treated with vitamin K,
a single dose of prednisone (2 mg/kg SC), and cephalexin
(20 mg/kg PO q8h). Considerable improvement in his
mentation was noted. However, after 5 days, anorexia,
hypersalivation, vomiting, and liquid diarrhea occurred,
and he returned to the local veterinary clinic, where he was
hospitalized and treated with intravenous fluids. The following
day, obtundation and facial twitching developed, and Murphy
was referred for further diagnostics and treatment.
Other Medical History: Murphy had been coughing a week
before he became systemically ill, but this subsequently
resolved. He was treated for a roundworm infection at that
time, which was diagnosed by fecal flotation. Murphy was
obtained from a breeder in Oklahoma at 4 months of age. He
had always appeared “slow” to the owner, his appetite had
never been very good, and he seemed unthrifty. The other
puppies in the litter were apparently healthy. There was no
known exposure to toxins. The breeder administered his first
vaccines, and he was revaccinated for distemper, parvovirus,
adenovirus, parainfluenza virus, and Leptospira by the local
veterinarian when he was acquired.
Physical Examination:
Body Weight: 12.2 kg.
General: Obtundation was noted; the dog was in lateral
recumbency and apparently unaware of his surroundings.
Facial twitching occurred throughout the examination.
PDH.20 Complications of pulmonary histoplasmosis include
granulomatous mediastinitis (characterized by enlargement and
sometimes calcification of mediastinal lymph nodes, with com-
pression of adjacent structures) or mediastinal fibrosis (char-
acterized by fibrosis of caseous mediastinal lymph nodes). The
clinical picture of PDH in humans is similar to that described in
dogs; rare clinical manifestations include meningitis, endocar-
ditis, and vascular infections.20,49 Humans with AIDS or those
treated with immunosuppressive drugs (such as tumor necrosis
factor antagonists) are at risk for severe PDH, and smokers are
predisposed to chronic pulmonary histoplasmosis. Itraconazole
prophylaxis is considered for immunosuppressed patients who
are at risk for development of the disease.20
Transmission of H. capsulatum from dogs or cats to humans
has not been reported, but disease in dogs and cats may signal the
potential for human infection as a result of exposure to the same
source of infection; outbreaks have been described that involved
both humans and dogs.60 The bodies of deceased pets with his-
toplasmosis should be disposed of promptly and by cremation.
T = 102.2°F (39.0°C), HR = 140 beats/min, RR = 50 breaths/min,
mucous membranes were moist and pink.
Eyes, Ears, Nose, and Throat: Moderate conjunctivitis was
present. There was a mild bilateral serous nasal discharge.
No other abnormalities were detected.
Musculoskeletal: Appeared small in stature, BCS 3/9.
Cardiovascular, Respiratory and Lymph Nodes: No clinically
significant abnormalities were detected.
Abdominal Palpation: Cranial organomegaly was present.
Laboratory Findings:
Blood Glucose: 70 mg/dL.
Plasma Fasting Ammonia Concentration: 509 µg/dL
(0-92 µg/dL).
Canine Distemper Virus Direct Immunofluorescent   Anti­
body (Urine Sediment): Negative.
CBC:
HCT 30.2% (40%-55%)
MCV 61 fL (65-75 fL)
MCHC 36.1 g/dL (33-36 g/dL)
Reticulocytes 49,200/µL (7000-65,000/µL)
WBC 22,000 cells/µL (6000-13,000 cells/µL)
Neutrophils 10,340 cells/µL (3000-10,500 cells/µL)
Band neutrophils 7040 cells/µL
Metamyelocytes 880 cells/µL
Lymphocytes 1100 cells/µL (1000-4000 cells/µL)
Monocytes 1100 cells/µL (150-1200 cells/µL)
Eosinophils 880 cells/µL (0-1500 cells/µL)
Platelets 28,000/µL (150,000-400,000 platelets/µL).
Moderately toxic neutrophils and markedly toxic band neu-
trophils and metamyelocytes were present. Occasional
organisms with morphology consistent with Histoplasma
capsulatum were seen within both neutrophils and mono-
cytes. The myeloid series was shifted to the myelocyte stage
with rare progranulocytes noted. Rare nucleated erythro-
cytes (metarubricytes) were also present. Many lympho-
cytes were reactive. Numerous macroplatelets were noted.
Serum Chemistry Profile:
Sodium 143 mmol/L (145-154 mmol/L)
Potassium 4.7 mmol/L (4.1-5.3 mmol/L)

Chloride 108 mmol/L (105-116 mmol/L)
Bicarbonate 18 mmol/L (16-26 mmol/L)
Phosphorus 7.7 mg/dL (3.0-6.2 mg/dL)
Calcium 10.3 mg/dL (9.9-11.4 mg/dL)
BUN 18 mg/dL (8-31 mg/dL)
Creatinine 0.4 mg/dL (0.8-1.6 mg/dL)
Glucose 117 mg/dL (70-118 mg/dL)
Total protein 5.8 g/dL (5.4-7.4 g/dL)
Albumin 2.1 g/dL (2.9-4.2 g/dL)
Globulin 3.7 g/dL (2.3-4.4 g/dL)
ALT 31 U/L (19-70 U/L)
AST 217 U/L (15-43 U/L)
ALP 195 U/L (15-127 U/L)
GGT 24 U/L (0-6 U/L)
Cholesterol 363 mg/dL (135-345 mg/dL)
Total bilirubin 1.1 mg/dL (0-0.4 mg/dL).
Urinalysis (Cystocentesis): SGr 1.044; pH 5.0, 1+ bilirubin,
no hemoprotein, no glucose, no protein, 0-1 RBC/HPF, rare
WBC/HPF; no crystals, casts, or bacteria seen.
Coagulation Panel: PT 10.7 s (7.5-10.5 s), PTT 24.4 s (9-12 s),
fibrin degradation products > 40 µg/mL (<10 µg/mL).
Imaging Findings:
Thoracic Radiographs: There was a diffuse bronchointerstitial
pulmonary pattern, especially in the region of the cranial
right middle lung lobe. The heart and pulmonary vasculature
were slightly small. In the viewable abdomen, the liver was
moderately enlarged.
Abdominal Ultrasound: Severe hepatomegaly was present
with normal hepatic echogenicity. A large, folded spleen and
sublumbar and mesenteric lymphadenomegaly were present.
Microbiologic Testing:
Fecal Flotation: Positive for Ancylostoma ova.
Aspiration Cytology (Ultrasound-guided Liver Aspirate):
The specimen was bloody and moderately cellular with
numerous clusters of platelets and inflammatory cells.
Occasional small clusters of hepatocytes were noted,
which showed mild to moderate vacuolar degeneration.
A few hepatocytes contained intracytoplasmic blue-green
pigment consistent with bile. Inflammatory cells consisted
primarily of activated macrophages that contained
abundant intracellular yeast organisms consistent with
Histoplasma spp. Lower numbers of nondegenerate
neutrophils, mixed lymphocytes, and occasional eosinophils
and basophils were also found. Interpretation: Histoplasma
infection with marked pyogranulomatous inflammation.
Mild to moderate hepatic vacuolar degeneration and
cholestasis.
CSF Analysis (Cisternal): The fluid was clear and colorless.
The protein concentration was 17 mg/dL (reference range,
<25 mg/dL). There was fewer than 1 RBC/µL (reference
range, <1200 RBC/µL) and 25 nucleated cells/µL (reference
range, <2 cells/µL). The differential cell count consisted
of 1% neutrophils, 6% lymphocytes, and 93% large
mononuclear cells. The cytofuge specimen was cellular,
and large, foamy macrophage-like cells predominated.
Vacuoles within the cytoplasm contained digested debris,
but no organisms could be discerned. Low numbers
of small lymphocytes were also noted. Interpretation:
Mononuclear pleocytosis.
Diagnosis: Acute progressive disseminated histoplasmosis with
suspected Histoplasma meningitis; hookworm infestation.
CHAPTER 61  Histoplasmosis 595
Treatment: Murphy was hospitalized in the intensive care
unit and treated with intravenous crystalloids (lactated
Ringer’s solution with 25 mEq/L KCl and 2.5% dextrose,
75 mL/h), plasma, vitamin K, and heparin (100 units/
kg SC q8h). The hookworm infection was treated with
fenbendazole (50 mg/kg PO q24h for 3 days). Seizures were
controlled with diazepam as needed and phenobarbital
(2 mg/kg IM q12h). Hepatic encephalopathy was treated
with a warm water enema followed by a 3 mL/kg lactulose
enema, then oral lactulose (1 mL/kg PO q8h). Profuse
watery diarrhea ensued. Treatment with lipid-complexed
amphotericin B was then initiated (2 mg/kg IV over 2 hours,
every other day). Initially, clinical signs improved, and
Murphy appeared brighter and began eating small amounts
of a reduced protein diet. A CBC showed a hematocrit of 30%,
19,988 neutrophils/µL, 263 band neutrophils/µL with slight
toxicity, 2367 lymphocytes/µL, 2367 monocytes/µL, 1315
eosinophils/µL, and 65,000 platelets/µL. No Histoplasma
organisms were seen. The results of a biochemistry profile
were similar to those reported at hospitalization, but
electrolytes and glucose had normalized and total bilirubin
was 2.6 mg/dL.
On day 4 of hospitalization, Murphy developed increased respi-
ratory effort, weakness, and a head tremor. Thoracic radio-
graphs showed a diffuse miliary interstitial infiltrate in the
perihilar region and in the region of the right cranial lung
lobe, which was slightly more severe than that seen at the
time of hospitalization. By day 7 of hospitalization, he re-
mained extremely weak and inappetent, and the owners
elected euthanasia because of financial limitations. At nec-
ropsy, there was severe hepatomegaly and splenomegaly;
the lungs were firm and mottled dark red to pink, and cut
surfaces oozed white froth and blood. Histopathology re-
vealed severe, diffuse, granulomatous inflammation with
intrahistiocytic yeasts in the lungs, liver, spleen, bone mar-
row, sublumbar lymph nodes, and tracheobronchial lymph
nodes (see Figure 61-8). In the kidneys there was severe,
multifocal, granulomatous interstitial nephritis with intra-
histiocytic yeasts. Multifocal granulomatous inflammatory
lesions with intrahistiocytic yeasts were present in the pan-
creas, duodenum, jejunum, thyroid glands, and peritoneal
fat. Within the brain there was minimal, focal, granuloma-
tous meningitis.
Comments: In this dog, the diagnosis of disseminated
histoplasmosis with fungemia was straightforward because
organisms were seen in a peripheral blood smear. The dog
likely acquired infection in Oklahoma where the fungus is
endemic. Overwhelming infection suggested a possible
underlying immunodeficiency, although severe disease
is common without obvious immunodeficiency in dogs.
The dog had evidence of disseminated intravascular
coagulation, hepatic encephalopathy, and granulomatous
meningitis. Had treatment with amphotericin B, azole
antifungal drugs, and additional supportive care continued,
recovery might have occurred, but the costs of this
treatment without guaranteed success led the owners
to elect euthanasia. Clearly the burden of intact fungal
organisms at necropsy was still massive despite several
treatments with amphotericin B. Even in human patients,
overall cure rates for H. capsulatum meningitis are no better
than 50%.
596 SECTION 3  Fungal and Algal Diseases
FELINE CASE EXAMPLE
Signalment: “Shaq,” an 8-month-old intact male domestic
shorthair cat from east central Louisiana.
History: Shaq was evaluated at the Louisiana State University
(LSU) Veterinary Medical Teaching Hospital for a 3-week
history of lethargy and intermittent anorexia. Occasional
right pelvic limb lameness had been noted. He was taken to
a local veterinary clinic, where fever and an enlarged right
popliteal lymph node were noted on physical examination.
A bite wound was suspected at the local veterinarian’s
clinic, and Shaq was treated with an injectable long-
acting cephalosporin and oral amoxicillin. After 2 weeks of
continued waxing and waning lethargy, Shaq was brought
to the emergency service at LSU.
Current Medications: Amoxicillin.
Other Medical History: Shaq was an indoor/outdoor cat
who has always lived in Louisiana. He ate a commercial
kitten food and was vaccinated at 3 to 4 months of age,
which included an FIV vaccination. FeLV and FIV infection
status was unknown.
Physical Examination:
Body Weight: 3.2 kg.
General: The cat was quiet but alert and responsive. His
mucous membranes were pink and moist, and his CRT was
less than 2 s. Rectal temperature was 104.9°F (40.5°C), and
heart rate was 216 beats/min.
Eyes, Ears, Nose, and Throat: Normal; no fundic examination
abnormalities were noted.
Musculoskeletal: Normal; no pain was noted on palpation of
the right pelvic limb. The right popliteal lymph node was
moderately enlarged, movable, and nonpainful.
Cardiovascular: No murmurs or arrhythmias were auscultated,
and a strong pulse quality was present.
Respiratory: Tachypnea was present but no significant
abnormalities were detected on auscultation.
Abdominal Palpation: Cranial organomegaly (splenomegaly)
was present.
Lymph Nodes: Other than the right popliteal lymph node,
lymph nodes were palpably normal.
Laboratory Findings:
CBC:
HCT 28.6% (30%-48%)
MCV 33 fL (41-57 fL)
MCHC 30.0 g/dL (30-35 g/dL)
Reticulocytes 22,100/µL
WBC 10,300 cells/µL (6000-10,000 cells/µL)
Neutrophils 6500 cells/µL (2500-12,500 cells/µL)
Lymphocytes 3700 cells/µL (1500-7000 cells/µL)
Monocytes 100 cells/µL (0-800 cells/µL)
Eosinophils 0 cells/µL (0-1500 cells/µL)
Platelets 515,000/µL (190,000-368,000
platelets/µL)
WBC and RBC morphology were considered normal.
Plasma protein 8.2 g/dL (6-7.5 g/dL).
Serum Chemistry Profile:
Sodium 164 mmol/L (150-165 mmol/L)
Potassium 3.7 mmol/L (4.0-5.0 mmol/L)
Chloride 126 mmol/L (121-126 mmol/L)
Phosphorus 6.5 mg/dL (3.1-7.5 mg/dL)
Calcium 10.3 mg/dL (8.8-9.3 mg/dL)
BUN 28 mg/dL (16-22 mg/dL)
Creatinine 1.1 mg/dL (1.7-1.9 mg/dL)
Glucose 125 mg/dL (77-96 mg/dL)
Total protein 7.8 g/dL (6.5-7.1 g/dL)
Albumin 3.0 g/dL (2.8-3.1 g/dL)
Globulin 4.8 g/dL (37.-4.1 g/dL)
ALT 77 U/L (24-44 U/L)
AST 16 U/L (0-36 U/L)
ALP 41 U/L (33-72 U/L)
Total bilirubin 0.2 mg/dL (0-0.4 mg/dL).
Urinalysis: Not performed.
Imaging Findings:
Thoracic Radiographs: There was a diffuse mild
bronchointerstitial pulmonary pattern that was somewhat
patchy in the caudodorsal lung fields. A smooth, broad-based
soft tissue opaque structure that was sharply marginated by
the surrounding lung was evident dorsal to sternebrae 3 to
4; this was consistent with sternal lymphadenopathy. In the
visible abdomen, the spleen was moderately enlarged (see
Figure 61-5, A).
Abdominal Ultrasound: A large spleen was present with
diffusely heterogenous echogenicity.
Microbiologic Testing:
Fecal Flotation: Negative.
FeLV/FIV ELISA Serology: Negative for FeLV antigen, positive
for FIV antibody.
Aspiration Cytology (Popliteal Lymph Node Aspirate):
Smears contained large numbers of adequately preserved
nucleated cells with a low number of erythrocytes. The
nucleated cell population consisted of high numbers of
small lymphocytes (75%), low numbers of neutrophils
(20%), and highly vacuolated macrophages (5%). The
macrophages contained large numbers of oval, 2- to 3-µm–
diameter yeast that were surrounded by a thin clear halo
and had small, eccentric, crescent-shaped purple nuclei.
Interpretation: Pyogranulomatous inflammation with
intracellular Histoplasma spp. yeasts (see Figure 61-7, B).
Histoplasma Quantitative Antigen EIA (Urine): 9.3 ng/mL
(positive).
Diagnosis: Disseminated histoplasmosis.
Treatment: Shaq was hospitalized in the intensive care unit
and treated with intravenous crystalloids (Normosol-R with
25 mEq/L KCl, 8 mL/h) and his temperature was monitored.
The fungal infection was treated with itraconazole oral
solution (10 mg/kg PO q24h). His temperature fluctuated
between 103.6°F and 105.3°F (39.8°C and 40.7°C) over the
next 48 hours, but he maintained a good appetite and
was bright and alert. On day 3, Shaq was discharged from
the hospital with instructions to continue the itraconazole
treatment. He was still febrile but was bright and alert and
eating well. Recommendations were to recheck monthly
at the local veterinary clinic for exam and chemistry
panel. A urinary Histoplasma Quantitative Antigen EIA was
repeated after 2 months of treatment and was 2.0 ng/mL,
which was still positive but indicated a probable treatment
response. Recheck at 6 months revealed a normal physical
examination, thoracic radiograph, CBC, and chemistry
panel with the exception of an increased serum ALT activity

at 221 U/L (0-90 U/L). It was recommended that the local
veterinarian repeat the Histoplasma antigen assay and
discontinue itraconazole if negative.
Comments: In this cat, a diagnosis of disseminated
histoplasmosis was straightforward because organisms were
seen in a lymph node aspirate. If they had not been present
on cytologic examination of the lymph node aspirate,
cytologic examination of a splenic or a bone marrow
aspirate would have been most likely to reveal organisms.
The vague presentation with mild respiratory signs is typical
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26. Lamm CG, Rizzi TE, Campbell GA, et  al. Pathology in prac-
tice. Histoplasma capsulatum infections. J Am Vet Med Assoc.
2009;235:155-157.
27. Vinayak A, Kerwin SC, Pool RR. Treatment of thoracolumbar
spinal cord compression associated with Histoplasma capsulatum
infection in a cat. J Am Vet Med Assoc. 2007;230:1018-1023.
28. Taylor AR, Barr JW, Hokamp JA, et al. Cytologic diagnosis of dis-
seminated histoplasmosis in the wall of the urinary bladder of a cat.
J Am Anim Hosp Assoc. 2012;48:203-208.
29. Kobayashi R, Tanaka F, Asai A, et al. First case report of histoplas-
mosis in a cat in Japan. J Vet Med Sci. 2009;71:1669-1672.
30. Burk RL, Jones BD. Disseminated histoplasmosis with osseous
involvement in a dog. J Am Vet Med Assoc. 1978;172:1416-1417.
31. Gilor C, Ridgway MD, Singh K. DIC and granulomatous vasculitis
in a dog with disseminated histoplasmosis. J Am Anim Hosp Assoc.
2011;47:e26-e30.
32. Lau RE, Kim SN, Pirozok RP. Histoplasma capsulatum infection in
a metatarsal of a dog. J Am Vet Med Assoc. 1978;172:1414-1416.
33. Nishifuji K, Ueda Y, Sano A, et  al. Interdigital involvement in a
case of primary cutaneous canine histoplasmosis in Japan. J Vet
Med A Physiol Pathol Clin Med. 2005;52:478-480.
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34. Kagawa Y, Aoki S, Iwatomi T, et al. Histoplasmosis in the skin and
gingiva in a dog. J Vet Med Sci. 1998;60:863-865.
35. Ueda Y, Sano A, Tamura M, et al. Diagnosis of histoplasmosis by
detection of the internal transcribed spacer region of fungal rRNA
gene from a paraffin-embedded skin sample from a dog in Japan.
Vet Microbiol. 2003;94:219-224.
36. Sano A, Miyaji M. Canine histoplasmosis in Japan. Nihon Ishinkin
Gakkai Zasshi. 2003;44:239-243.
37. Clemans JM, Deitz KL, Riedesel EA, et  al. Retroperitoneal pyo-
granulomatous and fibrosing inflammation secondary to fungal
infections in two dogs. J Am Vet Med Assoc. 2011;238:213-219.
38. Olson GA, Wowk BJ. Oral lesions of histoplasmosis in a dog. Vet
Med Small Anim Clin. 1981;76:1449-1451.
39. Huss BT, Collier LL, Collins BK, et al. Polyarthropathy and chorio-
retinitis with retinal detachment in a dog with systemic histoplas-
mosis. J Am Anim Hosp Assoc. 1994;30:217-224.
40. Gwin RM, Makley Jr TA, Wyman M, et  al. Multifocal ocu-
lar histoplasmosis in a dog and cat. J Am Vet Med Assoc.
1980;176:638-642.
41. Meadows RL, MacWilliams PS, Dzata G, et al. Diagnosis of his-
toplasmosis in a dog by cytologic examination of CSF. Vet Clin
Pathol. 1992;21:122-125.
42. Schaer M, Johnson KE, Nicholson AC. Central nervous system dis-
ease due to histoplasmosis in a dog: a case report. J Am Anim Hosp
Assoc. 1983;19:311-316.
43. Clinkenbeard KD, Cowell RL, Tyler RD. Disseminated histo-
plasmosis in cats: 12 cases (1981-1986). J Am Vet Med Assoc.
1987;190:1445-1448.
44. Carakostas MC, Miller RI, Gossett KA. Clinical laboratory
evaluation of deep mycotic diseases in dogs. J Small Anim Pract.
1984;25:687-693.
45. Kowalewich N, Hawkins EC, Skowronek AJ, et al. Identification
of Histoplasma capsulatum organisms in the pleural and peritoneal
effusions of a dog. J Am Vet Med Assoc. 1993;202:423-426.
46. Clinkenbeard KD, Cowell RL, Tyler RD. Identification of Histo-
plasma organisms in circulating eosinophils of a dog. J Am Vet
Med Assoc. 1988;192:217-218.
47. Burk RL, Corley EA, Corwin LA. The radiographic appearance of
pulmonary histoplasmosis in the dog and cat: a review of 37 case
histories. J Am Vet Radiol Soc. 1978;19:2-7.
48. Miller BM, Waterhouse G, Alford RH, et al. Histoplasma infection
of abdominal aortic aneurysms. Ann Surg. 1983;197:57-62.
49. Kauffman CA. Histoplasmosis: a clinical and laboratory update.
Clin Microbiol Rev. 2007;20:115-132.
50. Swartzentruber S, Rhodes L, Kurkjian K, et al. Diagnosis of acute
pulmonary histoplasmosis by antigen detection. Clin Infect Dis.
2009;49:1878-1882.
51. Wheat J, Wheat H, Connolly P, et  al. Cross-reactivity in Histo-
plasma capsulatum variety capsulatum antigen assays of urine
samples from patients with endemic mycoses. Clin Infect Dis.
1997;24:1169-1171.
52. Kuberski T, Myers R, Wheat LJ, et al. Diagnosis of coccidioidomy-
cosis by antigen detection using cross-reaction with a Histoplasma
antigen. Clin Infect Dis. 2007;44:e50-e54.
53. Hage CA, Kirsch EJ, Stump TE, et al. Histoplasma antigen clear-
ance during treatment of histoplasmosis in patients with AIDS
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Vaccine Immunol. 2011;18:661-666.
54. Cook AK, Cunningham LY, Cowell AK, et al. Clinical evaluation
of urine Histoplasma capsulatum antigen measurement in cats
with suspected disseminated histoplasmosis. J Feline Med Surg.
2012;14:512-515.
55. Martagon-Villamil J, Shrestha N, Sholtis M, et al. Identification of
Histoplasma capsulatum from culture extracts by real-time PCR.
J Clin Microbiol. 2003;41:1295-1298.
56. Babady NE, Buckwalter SP, Hall L, et  al. Detection of Blasto-
myces dermatitidis and Histoplasma capsulatum from culture isolates
and clinical specimens by use of real-time PCR. J Clin Microbiol.
2011;49:3204-3208.
57. Restrepo A, Tobon A, Clark B, et al. Salvage treatment of histo-
plasmosis with posaconazole. J Infect. 2007;54:319-327.
58. Wheat LJ, Connolly P, Smedema M, et al. Activity of newer triazoles
against Histoplasma capsulatum from patients with AIDS who
failed fluconazole. J Antimicrob Chemother. 2006;57:1235-1239.
59. Kroetz DN, Deepe GS. The role of cytokines and chemokines in
Histoplasma capsulatum infection. Cytokine. 2012;58:112-117.
60. Davies SF, Colbert RL. Concurrent human and canine his-
toplasmosis from cutting decayed wood. Ann Intern Med.
1990;113:252-253.
CHAPTER 62
Cryptococcosis
Jane E. Sykes and Richard Malik
Overview of Cryptococcosis
First Described: 1894, in a human patient in Germany (inde-
pendently by Busse and Buschke)1
Causes: Cryptococcus gattii, Cryptococcus neoformans, rarely
other cryptococcal species (a basidiomycete, teleomorph
Filobasidiella spp.)
Affected Hosts: Cats, dogs, humans, and a variety of other
domestic and wild animal species
Geographic Distribution: Worldwide
Major Mode of Transmission: Inhalation of basidiospores
from the environment
Major Clinical Signs: Nodular or ulcerative cutaneous lesions,
upper respiratory tract signs, chorioretinitis, neurologic
signs secondary to meningoencephalitis. In dogs, Cryp-
tococcus can also disseminate to abdominal organs and
cause gastrointestinal signs such as vomiting, diarrhea,
and inappetence.
Differential Diagnoses: For cats, differential diagnoses for
cutaneous lesions include other deep mycoses (especially
sporotrichosis), mycobacteriosis, nocardiosis, neoplasia
(especially squamous cell carcinoma), herpetic derma-
titis and eosinophilic granuloma complex. Differential
diagnoses for suspected cryptococcal rhinitis include
nasal lymphoma, foreign bodies, sinonasal/sino-orbital
aspergillosis, oronasal fistulae and neutrophilic rhinitis.
For ocular and central nervous system lesions, differential
diagnoses include neoplasia (especially lymphoma), toxo-
plasmosis, feline infectious peritonitis, and otogenic bac-
terial meningitis. For dogs, differential diagnoses include
other disseminated mycoses (especially histoplasmo-
sis and blastomycosis), mycobacteriosis, actinomycosis
and nocardiosis, other causes of rhinitis (such as foreign
bodies, nasal aspergillosis, oronasal fistulas, lymphoplas-
macytic rhinitis, rhinosporidiosis), neoplasia (especially
round cell neoplasia), toxoplasmosis, neosporosis, severe
inflammatory bowel disease, and pancreatitis.
Human Health Significance: Human infections occur as a
result of exposure to organisms in the environment and are
often associated with underlying immune compromise.
Etiology and Epidemiology
Cryptococcosis is an important fungal infection of people and
animals worldwide and the most common systemic mycosis
of cats. The infection is acquired from the environment, and
disease transmission from one animal to another has not been
reported. Occasional outbreaks of cryptococcosis in people
and animals may result from exposure to common environ-
mental sources.2,3 Various domestic and native mammals may
be infected, which include cats, dogs, ferrets, horses, camelids,
goats, sheep, cattle, dolphins, birds, koalas, and other marsu-
pials.4-10 The incidence of cryptococcosis in cats is eightfold
higher than that in dogs.11
The genus Cryptococcus contains at least 19 encapsulated
yeast species. Cryptococcus neoformans and Cryptococcus gat-
tii (formerly C. neoformans var. gattii) cause the vast majority
of infections in dogs, cats, and humans. C. gattii has emerged as
a pathogen of immunocompetent humans in temperate regions
of North America, in particular on the west coast of the United
States and in British Columbia, Canada. Other species, particu-
larly Cryptococcus laurentii and Cryptococcus albidus, rarely
cause disease in people and animals, but may be emerging as
a result of immunocompromise.12-14 Cryptococcus magnus
was isolated from the external ear canal of a cat with otitis
externa,15 and C. laurentii has been isolated from the external
ear canal of dogs.16
Cryptococcus spp. are dimorphic, basidiomycetous fungi.
The life cycle of the fungus is thought to involve growth in host
tissues as an encapsulated haploid budding yeast, combined
with the ability to undergo a transition to a filamentous form
(or teleomorph) in the environment. Two mating types of Cryp-
tococcus spp. exist, α and a. The α-type is vastly more preva-
lent in environmental and clinical samples. Under appropriate
laboratory conditions, the two mating types fuse and adopt a
dikaryotic filamentous state, also known as the perfect state.
This is followed by production of basidia, which are small, club-
shaped structures on which basidiospores form (Figure 62-1).17
Cells of the α mating type can also undergo asexual reproduc-
tion via a process known as monokaryotic fruiting, which also
involves filamentation and spore formation.17-19 Although they
have never been observed in nature, basidiospores (or possibly
desiccated yeasts) are thought to be the main form that, when
inhaled, gives rise to mammalian disease.20 In animal tissues,
basidiospores convert to the yeast form, which is round to oval
with a variably sized capsule. The capsule is primarily (90% to
95%) composed of a polysaccharide known as glucuronoxy-
lomannan (GXM), with smaller amounts of galactoxyloman-
nan. The capsule enlarges after infection of mammalian hosts
and protects the fungus from phagocytosis and environmental
insults such as desiccation. Within tissues, Cryptococcus repro-
duces by forming one or two daughter cells (buds) that are con-
nected to the parent cell by a narrow base (Figure 62-2).
Historically, five serotypes of Cryptococcus (A, B, C, D, and
AD) were recognized on the basis of antigenic differences in
the capsular polysaccharide. Based on the results of molecular
599
600 SECTION 3  Fungal and Algal Diseases

A B
FIGURE 62-1  Life cycle of Cryptococcus spp. A, Spores result from either mating between cells of opposite mating types (MATa or MATα) and subsequent formation of dikaryotic
hyphae or unisexual mating of MATα cells (monokaryotic fruiting). In both cases, sexual development within the filamentous form results in meiosis and basidiospore formation. The spores
germinate to produce haploid, yeast-like cells that divide by budding. B, Spores, and potentially desiccated yeast cells, are inhaled. Germination of spores or vegetative growth of yeast cells
in the upper or lower respiratory tract results in the proliferation of budding yeasts. Dissemination to a variety of tissues can then occur, but especially to the lymph nodes, nasal cavity, skin,
and CNS. (Modified from Kronstad JW, Attarian R, Cadieux B, et al. Expanding fungal pathogenesis: Cryptococcus breaks out of the opportunistic box. Nat Rev Microbiol 2011;9:193-203.
Basidiospore image courtesy Patrik Inderbitzen, University of California, Davis.)

cats have been predisposed in Australian studies but were not

FIGURE 62-2  CSF cytology from an 8-year-old male neutered domestic shorthair cat
with cryptococcal meningitis. Large numbers of budding cryptococcal yeasts are present.
typing methods such as PCR fingerprinting and multilocus
sequence typing, cryptococcal strains from around the world
are divided into eight major molecular types: VNI and VNII
(C. neoformans serotype A); VNIII (C. neoformans serotype
AD); VNIV (C. neoformans serotype D), and VGI, VGII, VGIII,
and VGIV (C. gattii, serotypes B and C).21 Hybrid strains also
exist. Differences in epidemiology, pathogenicity, clinical fea-
tures, and drug susceptibility have been associated with each
species and molecular type.
Cats of all ages may be affected,22 but young adult cats
appear to be at increased risk. In dogs, cryptococcosis is diag-
nosed predominantly in young adult dogs. In one study, the
median age of cats and dogs was 6 and 4 years, respectively.11
No clear sex predisposition exists. Siamese, birman, and ragdoll
found to be at risk in the United States.11,23 Similarly, German
shepherd dogs, Doberman pinschers, and great Danes appear to
be predisposed in Australia, whereas in the United States, Amer-
ican cocker spaniel dogs are strongly predisposed.5,11,22,24,25
Most cats and dogs with cryptococcosis do not have iden-
tifiable underlying immunosuppressive illness. The prevalence
of retrovirus infection in cat populations with cryptococcosis
does not differ from that in cat populations without crypto-
coccosis.11,22 However, some cats with cryptococcosis that are
co-infected with FeLV appear to respond more slowly to treat-
ment and may be more likely to have relapses.26 Cryptococcosis
is occasionally reported in cats treated with immunosuppres-
sive therapy or chemotherapy for malignancy.11,27 Concurrent
opportunistic infections have been reported in other cats (e.g.,
toxoplasmosis), as well as in dogs (e.g., neosporosis or papillo-
matosis), suggesting underlying immunodeficiency.11,28
Cryptococcus neoformans
C. neoformans has a worldwide distribution. It primarily infects
human patients who are immunocompromised, especially AIDS
patients. The realized environmental niche for C. neoformans
is weathered bird (especially pigeon) guano, in which it mates
prolifically.29 It can also be found in decaying plant matter in
hollows of certain trees.30 The organism can remain viable for
at least 2 years in environments such as pigeon lofts. The major-
ity of human cryptococcal infections result from infection with
C. neoformans var. grubii (VNI). C. neoformans var. grubii is
the most common Cryptococcus species isolated from dogs and
cats in southeastern Australia and accounts for more than 70%
of infections.22,23,25 Across the United States, most dogs with
cryptococcosis are infected with C. neoformans, but infection
of cats with this species is rare.11,31

Cryptococcus gattii
C. gattii is endemic in Australia, New Zealand, Papua New
Guinea, Southeast Asia, parts of Latin America, California, Mex-
ico, Hawaii, Central and South Africa, and, to a lesser extent,
certain parts of Europe (Austria, Germany, France, Italy, Greece,
and Spain).32,33 Since 1999, an epidemic of infections with
C. gattii has occurred in humans and domestic and wild ani-
mals in British Columbia in Canada, especially Vancouver
Island, and the Pacific Northwest of the United States.34-36
The main molecular types identified in this epidemic have been
molecular types VGIIa and, to a much lesser extent, VGIIb
and, in Oregon, also VGIIc. C. gattii molecular type VGII
can be found on living tree bark (Garry oak, maple, cedar,
and pine) and in air, and has also been found in freshwater
and seawater. Proximity to areas with soil disturbance was a
risk factor for dogs and cats.37 In California, almost all cats
are infected with C. gattii VGIII, with occasional VGII infec-
tions.11,31 The ecologic niche of this molecular type in the
United States remains unclear. Approximately 25% of affected
cats are housed exclusively indoors.11
In eastern Australia, 20% to 30% of human, feline, and
canine cryptococcosis is caused by C. gattii,23,38 whereas in
western Australia, approximately 50% of cats and dogs are
infected with C. gattii.5 Most C. gattii isolates from eastern Aus-
tralia belong to VGI; VGII predominates in southwestern and
northern Australia.5,39 C. gattii VGI in Australia is exclusively
associated with dead plant material in eucalyptus tree hollows.
Cats in rural environments are at risk of infection.
In contrast to C. neoformans, C. gattii isolates from the
Pacific northwest and British Columbia can infect apparently
immunocompetent humans.40 C. gattii molecular type VGIII
infections have been reported in immunosuppressed humans in
southern California.41 When disease occurs in immunocompe-
tent hosts, it is often characterized by the presence of pulmonary
mass lesions (cryptococcomas) and sometimes intracranial cryp-
tococcomas.42 As a result, pulmonary cryptococcomas are more
commonly described in human patients infected with C. gattii
compared with those infected with C. neoformans.43
Clinical Features
Signs and Their Pathogenesis
Based on the small size of basidiospores, the lung is thought to
be the primary site of infection in humans. In cats, dogs, and
koalas, the nasal cavity may be the primary site of infection (see
Figure 62-1); significant pulmonary parenchymal involvement
in these species is rare. Colonization of the nasal cavity may be
followed by development of cryptococcal rhinitis in some (but
not all) dogs and cats. However, some nasal cavity involvement
may result from hematogenous dissemination, because injec-
tion of Cryptococcus spp. into the carotid artery of cats can
lead to development of lesions on the bridge of the nose. Some
localized cutaneous lesions may result from direct cutaneous
inoculation (e.g., after a cat scratch), but skin lesions are usu-
ally interpreted as the result of dissemination or extension from
the nasal cavity.
The pathogenesis of cryptococcosis depends on the size of
the inoculum, virulence of the cryptococcal strain, and host
immunity. Cryptococcus has been aptly described as “a sugar-
coated killer with designer genes.”44 Cryptococcus has several
important virulence factors, including its polysaccharide cap-
sule, laccase (which makes melanin), and other enzymes such as
CHAPTER 62  Cryptococcosis 601
FIGURE 62-3  Ultrasound image that shows an enlarged and hypoechoic pancreas
in a 7-year-old female spayed basset mixed-breed dog with systemic cryptococcosis
caused by Cryptococcus neoformans var. grubii (molecular type VNI). The dog was evalu-
ated for gastrointestinal signs that were associated with severe cryptococcal pancreatitis
and enteritis. The dog’s lungs, heart, kidneys, liver, mesenteric lymph nodes, peritoneum,
brain, and meninges were also involved at necropsy.
phospholipase, urease, and superoxide dismutase.17,42 Capsular
polysaccharide is continuously shed into the host’s extracellular
fluid, which includes blood, urine, and CSF. The capsule inhib-
its phagocytosis, depletes complement, and is well known for
its ability to inhibit effective T cell responses. The ability of C.
neoformans and C. gattii to grow at mammalian body tempera-
ture is also an important virulence factor and is lacking in other
species of Cryptococcus.
The incubation period can range from 2 to 13 months.45,46
Exposure and self-limiting infection may occur in some individu-
als, with reactivation of viable cryptococci in residual granulo-
matous foci months or years later with immunosuppressive drug
therapy or illness.42 Because of this delay, a travel history to areas
where Cryptococcus is highly endemic (such as British Columbia
or the west coast of the United States) may be present in animals
and humans residing in non-endemic regions. After infection is
established within the lung or nasal cavity, the organism can
spread hematogenously to other sites, particularly the lymph
nodes, eye, skin, and central nervous system (CNS) (see Figure
62-1). Cryptococcus spp. has a strong tendency to invade the
meninges and typically causes meningitis or meningoencephali-
tis. In humans, cerebral cryptococcosis is often associated with
increased intracranial pressure, and this probably also occurs
in cats and dogs. Spread of infection to the eye, either along
the optic nerves from the brain or as a result of hematogenous
spread, may result in concurrent cryptococcal optic neuritis and
chorioretinitis. In animals with nasal cavity involvement, exten-
sion of infection into the brain may also follow osteomyelitis of
the cribriform plate or of the ventral wall of the frontal sinus.
Middle ear involvement can also occur.47 Infection also often
spreads from the nasal cavity to the mandibular lymph nodes.
Multifocal cutaneous involvement in cats and dogs reflects
hematogenous dissemination from the primary site of infection,
as do lesions in bone (e.g., digits) or periarticular soft tissues.
Dogs infected with C. neoformans often develop widely dis-
seminated disease, with involvement of sites such as the gastro-
intestinal system, pancreas, mesenteric lymph nodes, kidneys,
myocardium, and thyroid gland (Figure 62-3).11,24,48,49 In con-
trast, dogs infected with C. gattii molecular type VGII in the
602 SECTION 3  Fungal and Algal Diseases

United States tend to have isolated caudal nasal cavity crypto-
coccomas, which can extend through the cribriform plate into
the brain (Figure 62-4).
Clinical Features in Cats
Cryptococcosis is often a chronic infection in cats, and affected
cats are usually otherwise well but occasionally have mild
lethargy and inappetence. Fever is rare, and when it occurs,
it tends to be low-grade. Upper respiratory tract signs due to
nasal cavity involvement are common, sometimes with associ-
ated mandibular lymphadenopathy. Single or multifocal ulcer-
ated or nonulcerated cutaneous masses may also be present
(Figure 62-5).
Lower respiratory tract signs such as tachypnea are uncom-
mon but can occur as a result of pleural effusion or severe medi-
astinal lymphadenomegaly. Neurologic signs vary depending
on the location of lesions in and around the CNS (see Physical
Examination Findings), and, rarely, cats with CNS involvement
lack obvious neurologic signs. Optic neuritis and chorioretinitis
develop in as many as one third of affected cats and almost
always indicate CNS involvement.
Other signs include peripheral lymphadenomegaly (which
may be unassociated with skin lesions and is often asymmet-
ric), lameness due to osteomyelitis or cryptococcal arthritis, and
swollen digits. Rarely, involvement of the abdominal lymph
nodes, kidneys, spleen, liver, thyroid, and salivary glands occur.

B C
FIGURE 62-4  Caudal nasal cryptococcosis in dogs infected with Cryptococcus gattii. A, CT scan showing a large caudal nasal cavity cryptococcoma at necropsy that invaded the cribriform
plate and retrobulbar space in a 5-year-old Doberman pinscher infected with C. gattii molecular type VGIIa. There is a palisading periosteal response along the lateral margin of the right frontal and
lacrimal bones. B and C, Postcontrast magnetic resonance images of the head of a 2-year-old intact male malamute with a 1-week history of abnormal behavior and seizures. Cryptococcus gattii
was isolated from the nasal cavity. A large, contrast-enhancing mass is present in the caudal nasal cavity that invades the cribriform plate and extends into the brain. (Courtesy Dr. Stacey Hoffman.)

Clinical Features in Dogs
In contrast to cats, dogs frequently develop severe dissemi-
nated disease. In one study, 80% of dogs had involvement of
multiple anatomic sites, and 50% of dogs had involvement
of sites other than the nasal cavity, skin, lungs, lymph nodes,
kidney, eye, and CNS.11 Weight loss, lethargy, and inappe-
tence are common nonspecific findings. Rhinosinusitis from
cryptococcosis may be subclinical in dogs, so its incidence
may be underestimated.11,25 However, sneezing and muco-
purulent nasal discharge can also occur. Neurologic signs
FIGURE 62-5  Multiple ulcerated skin lesions in a cat from northern California with
disseminated cryptococcosis caused by Cryptococcus gattii molecular type VGIII.
A B
FIGURE 62-6  Ocular abnormalities in cats with disseminated cryptococcosis. A, Bilateral mydriasis in an 8-year-old male neutered domestic shorthair with cryptococcal menin-
goencephalitis. Fundoscopic examination was within normal limits. B, Granulomatous chorioretinitis and retinal detachment in another cat with cryptococcosis. (Courtesy University of
California, Davis, Veterinary Ophthalmology Service.)
CHAPTER 62  Cryptococcosis 603
were present in two thirds of affected dogs from ­California.11
Dogs can also primarily show signs of gastrointestinal or
pancreatic involvement, such as vomiting, diarrhea, and
abdominal pain.
Cutaneous involvement is uncommon in dogs but, as in cats,
may be a marker for disseminated disease. Although rarely
detected antemortem, more than half of dogs have lower respi-
ratory involvement at necropsy.11 Other clinical signs that are
infrequently present in dogs include peripheral lymphadeno-
megaly or lameness (due to osteomyelitis or arthritis).
Physical Examination Findings
Cats
Upper respiratory tract signs are common physical examina-
tion findings in cats with cryptococcosis. These include sneez-
ing; snuffling; and mucopurulent, serous, or hemorrhagic
nasal discharge that is unilateral or bilateral. In some cats, a
fleshy mass protrudes from the nostril. Other cats have one or
more firm to fluctuant, ulcerated or nonulcerated, subcutane-
ous swellings over the bridge of the nose. These swellings may
communicate with the frontal sinus or nasal cavity, and palpa-
tion of the lesions may reveal crepitus due to subcutaneous
emphysema. Cats with nasopharyngeal cryptococcosis develop
stertor, stridor, and, occasionally, otitis media. Mandibular
lymphadenopathy is often present. Ulcerated or proliferative
lesions of the buccal mucosa can also be found. Multifocal skin
lesions consist of nodules or masses that are fluctuant to firm,
up to several centimeters in diameter, and may ulcerate (see
Figure 62-5).
Neurologic examination abnormalities in cats with CNS
involvement include obtundation; atypical behavior; slow
menace, palpebral, or pupillary light responses; mydriasis
or anisocoria (Figure 62-6, A); twitching or tremors; and sei-
zures, circling, head pressing, ataxia, paresis, abnormal placing
reactions, head tilt, nystagmus, and spinal pain.9 Fundoscopic
examination may reveal granulomatous chorioretinitis, exuda-
tive retinal detachment, papilledema, evidence of optic neuri-
tis, and/or retinal hemorrhage (Figure 62-6, B). Severe ocular
604 SECTION 3  Fungal and Algal Diseases

TABLE 62-1
Diagnostic Assays Currently Available for Cryptococcosis in Dogs and Cats
Assay Specimen Type Target Performance
Cytologic Aspirates of affected tissues, Cryptococcus High sensitivity, but occasionally organisms are not
­examination body fluids, impression ­organisms ­visualized (such as in the CSF of some animals with
smears of skin lesions or CNS cryptococcosis).
biopsies
Fungal culture Aspirates or biopsies of af- Cryptococcus spp. Generally sensitive and specific, but false negatives are
fected tissues, body fluids possible if specimen size is low. Requires several days’
such as CSF and urine incubation. Allows species identification and antifungal
drug susceptibility testing.
Antigen assay Serum, CSF Cryptococcus Sensitivity and specificity exceed 90%. False negatives
(latex agglu- ­polysaccharide can occur in dogs and with localized infections in cats.
tination) antigen False positives are generally of low magnitude (<1:200
and usually <1:16). Titers vary between kits.
Real-time PCR Biopsies, body fluids, aspi- Cryptococcus DNA Not yet validated in adequate numbers of dogs or cats
assays rates with cryptococcosis; usefulness requires further evalu-
ation.
Histopathology Biopsy specimens from af- Cryptococcus spp. High sensitivity, with rare false negatives. Special stains
fected tissues yeasts such as Mayer’s mucicarmine and immunohistochemi-
cal stains facilitate organism detection and identifica-
tion.

lesions or optic neuritis may result in blindness with dilated and
unresponsive pupils.
Dogs
Many dogs with cryptococcosis are lethargic or obtunded
because of disseminated disease and/or CNS involvement. When
fever occurs, it is typically low grade. Other CNS signs in dogs
include absent or slow menace, gag, pupillary light responses,
nystagmus, mydriasis, head tilt, strabismus, anisocoria, facial
paralysis, paraparesis, abnormal placing reactions, ataxia, cir-
cling, seizures, twitching or tremors, reluctance to open the
mouth, absent or decreased segmental reflexes, and, commonly,
cervical hyperesthesia.9,24 Some dogs show signs that resemble
those of cervical disc herniation. The most common ocular
abnormalities are chorioretinitis, papilledema, evidence of optic
neuritis, and retinal detachment. Decreased nasal airflow may
be present in dogs with nasal cavity cryptococcomas. Nodules,
masses, or ulcers may involve any portion of the integument, and
the nose, tongue, buccal mucosa, hard palate, lips, or nail beds.
Diagnosis
Definitive diagnosis of cryptococcosis is usually straightforward
and based on evaluation of representative tissue specimens with
cytology, culture, and occasionally histopathology, with or with-
out serologic detection of cryptococcal antigen in body fluids
(Table 62-1). Suitable specimens include nasal swabs, nasal wash-
ings, needle aspirates from mass lesions or enlarged lymph nodes,
bronchoalveolar lavage specimens, pleural fluid, CSF, and urine.
Laboratory Abnormalities
Complete Blood Count, Serum Chemistry Profile, and Urinalysis
Changes in the CBC, chemistry panel, and urinalysis in animals
with cryptococcosis are generally mild and nonspecific. Mild to
moderate nonregenerative anemia; leukocytosis, often with mono-
cytosis; and eosinophilia are the most common findings. Dogs with
disseminated disease may have a neutrophilia with a left shift. The
serum biochemical profile reflects specific organ involvement in
animals with disseminated disease. Occasionally, cryptococcal
yeasts are found on examination of the urine sediment.
Cerebrospinal Fluid Examination
The CSF in cats and dogs with neurologic signs due to crypto-
coccosis may have an increased protein concentration (usually
between 10 and 140 mg/dL in cats, but may be several hundred
or rarely more than 1000 mg/dL in dogs).9 Leukocyte counts
can range from 2 to more than 1000 cells/µL in both dogs and
cats. A mixed cellular pleocytosis is generally present, with
neutrophils or large mononuclear cells generally outnumbering
other cell types. Some dogs have a marked eosinophilic pleocy-
tosis. Cryptococcal yeasts are visible in most but not all animals
with cryptococcal meningitis (see Figure 62-2).9 Marked deteri-
oration in neurologic status occasionally occurs in animals with
CNS cryptococcosis immediately after CSF collection. Many
animals have intracranial mass lesions or increased CSF pres-
sure, which can increase the risk of cerebellar herniation fol-
lowing a cisternal tap. Wherever possible, attempts to make a
diagnosis with serum antigen testing or collection of specimens
from other anatomic sites should be considered as an alternative
to diagnosis by CSF analysis.
Diagnostic Imaging
Plain Radiography
Thoracic radiographs in dogs and cats are often normal. In
some animals, interstitial to alveolar infiltrates or small nodu-
lar lesions are present. Occasionally, large pulmonary nodules,
hilar lymphadenopathy, a mediastinal granuloma, or pleural
effusion are found.41
 CHAPTER 62  Cryptococcosis 605

FIGURE 62-7  Transverse T1-weighted pre-contrast (left), T1-weighted post-contrast (center), and T2-weighted (right) MRI of the brain of an 8-year-old male neutered domestic short-
hair with cryptococcosis. There is a focal lesion in the gray matter of the right cerebral cortex at the junction of the parietal and occipital lobes (arrowhead). The lesion is T2 hyperintense, T1
hypointense, and rim-enhancing (arrow). These signal characteristics were consistent with a pseudocyst lesion.

Computed Tomography
In cats with nasal cryptococcosis, computed tomography (CT)
of the nasal cavity can reveal soft tissue and fluid opacifica-
tion of the nasal cavity or frontal sinus, peripherally contrast-
enhancing mass lesions of the nasal planum, and lysis of the
nasal bones or of the cribriform plate.11 In dogs, CT findings in
the nasal cavity include peripherally contrast-enhancing mass
lesions within the frontal sinuses, caudal nasal cavity, nasophar-
ynx, or the retrobulbar space and associated osteomyelitis (see
Figure 62-4, A). CNS abnormalities such as ventricular dila-
tion and intra-axial contrast-enhancing mass lesions have been
described in dogs using CT.9 However, MRI is more sensitive
for detection of CNS lesions in human patients,42 and the same
is probably true for dogs and cats.

Magnetic Resonance Imaging

MRI abnormalities in cats with CNS cryptococcosis consist of
single or multifocal contrast-enhancing mass lesions that tend
to be hyperintense on T2-weighted images and hypointense
on T1-weighted images, as well as mild to moderate, diffuse
or focal meningeal enhancement.9 Some cats have lesions that
appear fluid-filled on T2-weighted images but have a greater
T1-weighted intensity than expected for acellular fluid (“high
T2, low T1 lesions”) (Figure 62-7).9 MRI in dogs may show
contrast-enhancing mass lesions that are hyperintense on
T2-weighted images and of variable intensity on T1-weighted
images, as well as meningeal enhancement (see Figure 62-4, B).
Ventriculomegaly and/or cribriform plate osteomyelitis may
also be evident.
Sonographic Findings
Over 80% of cats with cryptococcosis have normal abdomi-
nal ultrasound examinations. Cats with kidney involvement
can have iso- to hypoechoic intraparenchymal mass lesions
on ultrasound, with or without associated renal failure. These
sometimes involve the renal pelvis. However, cats with renal
involvement can also have normal abdominal ultrasound
examinations.11
Ultrasonographic findings in dogs include intra-­abdominal
lymphadenomegaly, multifocal gastric and small intestinal thick-
ening, changes suggestive of pancreatitis, renal mass lesions,
and/or ascites (see Figure 62-3).11,48
FIGURE 62-8  India ink stained preparation of Cryptococcus gattii yeasts that had
been grown in culture. 1000× oil magnification. (Courtesy Greg Hodges and George
Thompson III, University of California, Davis.)
Microbiologic Tests
Cytologic Examination
Cytologic examination can be performed on aspirates, body flu-
ids, or impression smears of biopsy specimens. Romanowsky-
type stains, new methylene blue, and Gram stain are all
satisfactory for making a cytologic diagnosis. India ink can be
used to examine CSF for cryptococcal yeasts, which appear
unstained and silhouetted against a black background. India ink
staining of CSF is still used routinely in the diagnosis of human
cryptococcal infections (Figure 62-8).42 However, lymphocytes
and fat droplets can sometimes be confused with the organism.
Although cytologic examination of nasal or cutaneous exu-
dates, aspirates, or body fluids for organisms is a rapid and
sensitive test for cryptococcosis, negative test results do not
eliminate the possibility of cryptococcosis. Generally, organ-
isms are found only by cytologic examination of CSF in 60% to
80% of dogs and cats.11 If a sufficient index of suspicion exists,
606 SECTION 3  Fungal and Algal Diseases
additional diagnostic tests such as histopathology, antigen test-
ing, and/or fungal culture are indicated.
Fungal Culture
Cryptococcus spp. are readily cultured from aspirates, exudate,
CSF, urine, and tissue specimens. Attempts to culture the organ-
ism are recommended whenever possible before treatment is
initiated, because isolation confirms the diagnosis, can be used
to differentiate C. gattii from C. neoformans, and permits anti-
fungal susceptibility testing for cases that subsequently fail to
respond adequately to antifungal drug treatment. Some veteri-
nary diagnostic laboratories still report all Cryptococcus iso-
lates as C. neoformans in the absence of tests to discriminate
between C. neoformans and C. gattii. If the report lists C. neo-
formans as the isolate, the laboratory must therefore be con-
sulted to determine whether C. neoformans was differentiated
from C. gattii. However, whether the clinician needs to know
whether the isolate is C. neoformans or C. gattii is debatable
because at this time, species identification does not clearly influ-
ence treatment recommendations or prognosis. If Cryptococcus
is isolated from a contaminated site such as the nasal cavity, a
diagnosis of cryptococcosis should be supported with cytologic
or histopathologic evidence of active infection. This is because
subclinical colonization occurs in some animals.
C. neoformans and C. gattii grow on almost all laboratory
media, but fungal isolation media (such as Sabouraud’s dextrose
agar) and incubation temperatures of 25°C or 30°C in addition
to incubation at 37°C are preferred when fungi are being con-
sidered in the differential diagnosis. Antibiotics are added to the
medium for isolation of Cryptococcus spp. from contaminated
sites, such as the nasal cavity. Colonies usually are visible in 2 to
3 days, but sometimes incubation for up to 10 days is required.
Unlike other dimorphic fungi, Cryptococcus grows as a yeast,
rather than a mold, on routine fungal media and so is less likely
to represent a laboratory hazard than organisms that grow as
molds. The organisms form white, creamy colonies. Often
C. gattii colonies are more mucoid than C. neoformans colonies.
The organism is identified based on its microscopic morphology
and response to biochemical tests in commercial yeast identifica-
tion kits (see Chapter 4). Canavanine-glycine-bromothymol blue
(CGB) agar is used to differentiate between C. neoformans and
C. gattii.50 Unlike C. neoformans, C. gattii grows on CGB agar
and turns the media a brilliant deep blue (Figure 62-9). Fungal
culture of the CSF is frequently positive in patients with CNS
cryptococcosis and should be performed even if the organism
cannot be demonstrated cytologically or serologically. Culture
of urine specimens collected using cystocentesis is also indicated
when cryptococcosis is suspected, even when obvious renal
involvement is not present and cytologic abnormalities are not
present. Identification of molecular types for epidemiologic pur-
poses requires specialized techniques such as multilocus sequence
typing or PCR fingerprinting. However, matrix-assisted laser
desorption/ionization–time of flight mass spectrometry (MALDI-
TOF) can also rapidly differentiate between Cryptococcus spe-
cies as well as molecular types, and may be used in veterinary
clinical microbiology laboratories in the future.51
Most cryptococcal isolates from dogs or cats are susceptible
to amphotericin B, 5-flucytosine, and azoles. Rarely, resistance
to 5-flucytosine and azole antifungal drugs (especially flucon-
azole) is detected. In general, minimum inhibitory concentra-
tions (MICs) for fluconazole are higher than those for other
azole drugs, and this does not imply resistance in vivo. In the
FIGURE 62-9  Growth of C. neoformans (left) and C. gattii (right) on canavanine-
glycine-bromothymol blue agar. Cryptococcus neoformans does not grow well in the pres-
ence of canavanine and does not utilize glycine, so the medium color remains unchanged.
Cryptococcus gattii grows in the presence of l-canavanine and utilizes glycine as the sole
carbon and nitrogen source, which raises the pH of the medium and causes the bromothy-
mol blue indicator in the medium to turn the agar a vivid cobalt blue color.
human literature it has been suggested that when the MIC rises
during treatment, or if the initial MIC is at least 16 µg/mL for
fluconazole, or at least 128 µg/mL for flucytosine, failure of
antifungal drug treatment may result from drug resistance.42
Serologic Testing
Latex agglutination assays for detection of cryptococcal poly-
saccharide capsular antigen are widely used for diagnosis of
cryptococcosis in veterinary patients and are sensitive and
specific.11,52,53 ELISA assays are also available, and the perfor-
mance of a rapid lateral-flow assay (CrAg, IMMY Diagnostics,
Norman, OK) is under investigation in the authors’ labora-
tories. Serologic assays detect the antigen of all C. gattii and
C. neoformans molecular types and can be used on serum or
CSF. These can provide a rapid diagnosis when organisms
cannot be visualized or cultured. When positive, serum cryp-
tococcal antigen assays can be advantageous in animals with
neurologic signs when CSF collection is unacceptably risky or if
a diagnosis of cryptococcosis is unlikely but requires additional
evidence for exclusion.
In human patients, commercial kits typically have at least
90% sensitivity and specificity.42 Results have been similar for
small animal patients,11,52,53 although false negative results may
be more common in dogs than in cats.11 False positive test results
are reduced when test specimens are pretreated in the laboratory
with pronase (a proteinase).42 In human patients, titers below
1:8 can represent false-positive results, although false-positive
titers of up to 1:200 have rarely been detected in cats.11 These
cats had other causative etiologies identified that explained their
clinical signs. In some cats these low positive titers may repre-
sent subclinical colonization. Attempts to obtain cytologic or
histopathologic confirmation of infection in animals with titers
of 1:200 or less are recommended.
Antigen titers are frequently extremely high (>1:65,536) in
cats and dogs with cryptococcosis, but even a titer of 1:2 can
indicate cryptococcal infection.52 Titer results from different
kits vary considerably; therefore, the methodology should not
be altered when monitoring the response to treatment. A good
prognosis is indicated by a decrease in antigen titer, whereas

FIGURE 62-10  Histopathology showing the meninges of a cat with cryptococcosis. Numerous round yeast organisms occupy the meningeal spaces. H&E stain.
a persistent titer after treatment suggests continued infection.
Reductions in the titer typically lag behind clinical improve-
ment. No correlation has been found between pretreatment
antigen titer and outcome.
Because the sensitivity of cryptococcal antigen testing is not
100%, cryptococcosis should not be ruled out on the basis of
a negative antigen titer alone. This is especially important in
dogs, because false negatives are more common in dogs than in
cats.11 False negatives are most common in animals with local-
ized ocular, CNS, or solitary cutaneous lesions. For dogs and
cats suspected to have cryptococcal meningoencephalomyelitis
that have negative serum antigen titers, CSF should be submit-
ted for cytologic examination, antigen titers, and fungal culture.
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR assays for Cryptococcus spp. are offered on a
commercial basis by some veterinary diagnostic laboratories.
These assays can confirm the presence of Cryptococcus DNA in
tissues or body fluids when other means fail. However, because
a diagnosis of cryptococcosis is usually apparent based on the
methods described earlier, PCR is currently not regarded as a
routinely useful clinical tool. Because Cryptococcus DNA has
been detected using PCR assays in small intestinal contents
from healthy dogs and dogs with chronic enteropathies,54 posi-
tive results on intestinal biopsies may not reflect ­cryptococcosis.
Similarly, because subclinical nasal cavity colonization can
occur, positive results on nasal biopsies may not represent inva-
sive disease. Because false positives also have the potential to
occur as a result of contamination in the PCR laboratory, a
PCR diagnosis of cryptococcosis should always be supported by
cytologic or histopathologic evidence of infection and/or culture
of Cryptococcus spp. from a normally sterile site.
Pathologic Findings
Lesions associated with cryptococcosis vary from gelatinous
masses that consist almost exclusively of organisms to well-
ordered granulomas or pyogranulomas that typically occur in
patients with improved cell-mediated immune (CMI) responses.
The gelatinous appearance is a reflection of the large amount
of capsular polysaccharide present in lesions. In sections
CHAPTER 62  Cryptococcosis 607
stained with hematoxylin-eosin, Cryptococcus yeasts are faint,
eosinophilic, round to oval bodies that are surrounded by a
clear halo (the unstained capsule). The organism is more eas-
ily visualized with periodic acid–Schiff, methenamine silver,
or Fontana-Masson stain, although the capsule still does not
stain well with these methods. With Mayer’s mucicarmine
stain, the cryptococcal capsule takes on a rose-red color, and
the organism appears pink against a blue background. Other
fungi with similar morphologic features do not stain with
Mayer’s mucicarmine. Some Cryptococcus strains have poorly
developed capsules. If necessary, immunohistochemistry can
be used to differentiate Cryptococcus from other fungi and
some reagents can provide information on the identity of the
species and serotype present.55
Cats
In cats that die or are euthanized as a result of cryptococco-
sis, histopathology of lesions within the nasal cavity may reveal
granulomatous rhinitis. CNS cryptococcosis consists of either
meningitis41 or meningoencephalomyelitis, with single or mul-
tiple cerebral granulomas (Figure 62-10). Extension of infection
from the meninges along perforating arteries into the Virchow-
Robin spaces can result in cystic collections of yeast organisms
known as gelatinous pseudocysts.9 Ocular lesions include pyo-
granulomatous to granulomatous chorioretinitis, optic neuritis,
uveitis, endophthalmitis, panophthalmitis, and retinal separa-
tion. Other affected organs include skin and subcutaneous tis-
sues, kidneys, and lymph nodes. Renal granulomas have been
found in some cats with disseminated disease. Granulomas can
also occasionally be found in the spleen, adrenal glands, thyroid
glands, and liver.
Dogs
Dogs that die or are euthanized because of cryptococcosis often
have CNS infection, ocular involvement, or widely disseminated
disease. The lesions consist of granulomatous or pyogranulo-
matous meningoencephalitis, sometimes with pseudocyst for-
mation; neuritis of the optic, facial, or vestibular nerves; and
pyogranulomatous or granulomatous chorioretinitis. Inflam-
matory lesions that contain Cryptococcus yeasts may also be
608 SECTION 3  Fungal and Algal Diseases
present in the lungs, nasal cavity, kidneys, pancreas, liver, myo-
cardium, gastrointestinal tract, and spleen. Less commonly,
involvement of the peritoneum, thyroid, tongue, prostate,
pleura, joints, mediastinum, urinary bladder, and adrenal gland
has been detected at necropsy.
Treatment and Prognosis
Treatment recommendations for dogs and cats with cryptococ-
cosis have been based on evidence-based studies performed in
human patients and clinical experiences; evidence-based studies
are lacking for veterinary patients. The most common problems
encountered are the high cost of therapy, requirement for mul-
tiple hospital visits, and the need for regular medication of pets
for a protracted period. Immunocompromised animals (which
may include purebred dogs with no obvious underlying immu-
nodeficiency) are especially likely to have persistent or progres-
sive infection in the face of treatment. In many affected animals,
treatment must be continued for years or the life of the animal.
Surgical debulking could be considered before treatment for
large cryptococcomas to improve drug penetration. However,
even large cryptococcomas can resolve with medical treatment
alone, and surgery should only be performed if lesions can be
easily and safely resected. The reader is referred to Chapter 9 for
detailed information on antifungal drugs.
Cats
Cats with localized cutaneous disease can sometimes be treated
successfully with azole monotherapy. The initial drug of choice
is fluconazole (10 mg/kg PO q12h), because of its good penetra-
tion of the brain, eye, and urinary tract; low cost; and minimal
adverse effects.
Treatment should be continued until there is complete lesion
resolution and the cryptococcal antigen titer becomes negative.
A four- to fivefold reduction in the antigen titer suggests an
adequate response. In cats with C. gattii molecular type VGI or
C. neoformans infections, this typically takes 2 to 12 months
(median, 4 months),56 although longer periods of therapy may
be required for some cats. For recovered animals that achieve
an antigen titer of 0, the titer can be monitored every 3 to
6 months, so that any recurrence is diagnosed early. However,
in the authors’ experience, many cats with C. gattii molecular
type VGII and VGIII infections in North America fail to respond
completely to fluconazole treatment, antigen titers persist at high
levels, and relapse occurs after an initial treatment response.
In some of these cats, treatment with itraconazole (5-10 mg/
kg PO q24h [capsules] or 3-5 mg/kg PO q24h [solution]) or
ketoconazole can lead to clinical remission. Liver enzyme activi-
ties should be monitored monthly during treatment with azole
antifungal drugs to monitor for hepatotoxicity (see Chapter 9).
Amphotericin B is the most effective anticryptococcal
agent. Penetration of the CNS and vitreous is poor, but clini-
cal responses still occur in cats and dogs with CNS and ocular
cryptococcosis, presumably because the blood-brain barrier is
compromised. A combination of amphotericin B and 5-flucy-
tosine (250 mg PO q8h) is considered the optimal therapy for
cats (and humans, but not dogs) with CNS cryptococcosis, or
for those that fail to respond to azole drugs. Fluconazole can
be substituted for 5-flucytosine if 5-flucytosine is unaffordable.
However, 5-flucytosine shows synergy when combined with
amphotericin B and effectively penetrates the blood-brain bar-
rier. Because resistance can develop rapidly when 5-flucytosine
is used alone, it is used chiefly to improve the efficacy of other
antifungal drugs. Lipid-complexed amphotericin B prepara-
tions are not necessarily more effective but are less nephro-
toxic, which may translate into greater efficacy because higher
doses can be used, although at a substantially increased cost. In
human patients, higher doses of amphotericin B more effectively
sterilize the CSF. The reader is referred to Chapter 9 for dosing
protocols for various formulations of amphotericin B. Treat-
ment with 5-flucytosine (or fluconazole) and amphotericin B is
continued for 2 to 4 weeks (ideally 4 weeks) or until azotemia
develops, after which treatment is continued with azole mono-
therapy. Additional courses of amphotericin B may be required
to maintain clinical remission in some cats. Although controlled
trials have not been performed, some cats with CNS cryptococ-
cosis have shown apparent response to addition of terbinafine
to the treatment regimen, when other drugs failed to resolve
clinical signs.57
Dogs
If affordable to the owner, dogs with disseminated disease or
CNS involvement should be treated initially with deoxycho-
late or lipid-complexed amphotericin B and fluconazole. Dogs
treated with flucytosine frequently develop toxic epidermal
necrolysis, typically 10 to 14 days after starting therapy, which
precludes treatment with flucytosine in this species.58 Some
dogs with CNS involvement respond to azole monotherapy,
although residual neurologic signs may persist.9 Other potential
outcomes include incomplete recovery, with persistent active
infection despite therapy, or relapse after prolonged clinical
remission. In one dog, surgical debridement of an extradural
spinal cord granuloma combined with fluconazole treatment
controlled spinal cord infection.59 Again, azoles are fungistatic
and rely on a CMI response and phagocytosis to remove crypto-
coccal yeasts from the host, which may be lacking in some dogs
with cryptococcosis. Treatment of a dog with intestinal crypto-
coccosis with terbinafine was associated with long-term disease
resolution when treatment with azoles, surgical debulking, and
amphotericin B failed.48
Other Antifungal Drugs
Other antifungal drugs that are effective for treatment of cryp-
tococcosis include voriconazole and posaconazole, although
these can be unaffordable for many clients. Voriconazole pene-
trates the CNS and so is potentially advantageous for treatment
of cryptococcal meningoencephalitis. However, virtually all cats
and some dogs do not tolerate voriconazole treatment because
of gastrointestinal, neurologic, and cardiac adverse effects. In
humans, posaconazole is well tolerated, and it has been used to
treat other fungal infections without complication in cats (see
Chapter 9), so it could be an option for cats with refractory dis-
ease. Unpredictable serum levels have led to increased interest in
therapeutic serum level determination in humans.
Supportive Care
Because many cats and dogs with CNS involvement deterio-
rate clinically during initial treatment with antifungal drugs
as a result of the inflammatory response to dying yeasts, anti-­
inflammatory doses of short-acting glucocorticoids may be nec-
essary to support animals with CNS cryptococcosis for the first
few days to weeks of treatment and may increase survival rate
at 10 days after initiation of treatment.9 Deterioration of neu-
rologic status in the first few days of treatment is not a negative

prognostic indicator if the animal can be supported through this
phase. Supportive care with IV fluid therapy, tube feeding, and
antiseizure medications may be necessary. Rarely, dogs or cats
require supplemental oxygen because of respiratory involvement.
Prognosis
In general, cats show significant clinical improvement within
1 to 2 weeks of starting therapy. In studies from Australia,
around 75% of cats were treated successfully.5,56 As many as
60% of cats may be cured after an initial course of therapy,
although some cats that show an initial favorable response to
therapy can relapse, sometimes as long as 10 years after therapy
is discontinued.56 In one study, the median survival time of cats
with CNS cryptococcosis was only 13 days (range, 0 to 4050
days), but a median survival time was never reached if cats sur-
vived at least 3 days after diagnosis, so supportive care during
this phase may be critical.9
The prognosis is more guarded for dogs. Six of 11 dogs with
cryptococcosis in Australia were treated successfully.56 In a
study from the western United States9 and another from south-
western Australia,5 the success rate was lower, around 30%.
The median survival time for dogs with CNS cryptococcosis
was 7 days (range, 0 to 3680 days).9
In human patients with cryptococcosis, negative prognostic
indicators include underlying immunosuppressive disease or
drugs, abnormal mental state, high organism burden at presen-
tation (as defined by large numbers of yeasts in the CSF and/or
high antigen titers), and a poor anti-inflammatory response in
the CSF (<20 cells/µL).60 The presence of CNS disease appears
to negatively affect outcome in both cats and dogs.56,61 In dogs
and cats with CNS involvement, the presence of altered mental
status at first evaluation was associated with increased mortality
(P = 0.03).9
Immunity and Vaccination
Cryptococcosis can occur in apparently immunocompetent or
in immunodeficient individuals, although those with deficiencies
of cell-mediated immunity are most commonly infected. Pro-
duction of Th1 type cytokines such as Il-2, TNF-α, and IFN-γ
and a granulomatous inflammatory response are important for
elimination of infection, and decline in the CD4+ T cell count
CASE EXAMPLE
Signalment: “Simon,” an 8-year-old male neutered domestic
shorthair cat from northern California
History: Simon was evaluated by the University of California,
Davis, Veterinary Medical Teaching Hospital (VMTH) for
a 5-week history of progressive weakness, inappetence,
and weight loss. Initially the owners noted that he
was reluctant to jump and had a low tail carriage, and
intermittent episodes of head shaking and sneezing were
also noted. He also had episodes of clenching the furniture
with his claws. Three weeks before he was brought to the
VMTH, Simon was seen at a local veterinary clinic where a
physical examination was unremarkable. Broad-spectrum
antimicrobial drugs were prescribed (amoxicillin-clavulanic
CHAPTER 62  Cryptococcosis 609
to less than 50 to 100 cells/µL correlates well with the risk of
infection in HIV-infected humans.42 No vaccines are available.
Prevention
Prevention of cryptococcosis may be possible through avoid-
ance of exposure to ecologic niches of the organism, such as
pigeon feces. Nevertheless, cryptococcosis occurs in animals
housed exclusively indoors, and widespread exposure and
recovery likely occurs in endemic areas.
Public Health Aspects
Cryptococcosis is an important opportunistic fungal infection
of people with AIDS, in particular those who are not treated
with highly active antiretroviral therapy. Human infection is
acquired from the environment and not by direct contact with
infected animals, although intense exposure to avian excrement
has been associated with some cryptococcosis cases in HIV-
infected humans.62 Malignancy, chemotherapy, immunosup-
pressive drug therapy, diabetes mellitus, splenectomy, cirrhosis,
or sarcoidosis can also predispose to the disease. Severe menin-
gitis and disseminated infections can develop in immunodefi-
cient patients.
The organism does not aerosolize from sites of tissue infec-
tion, so the disease cannot spread among people or animals.
The major public health significance of infected pets is that
cats may act as a sentinel for infection of human beings,
because of their susceptibility to infection. In the diagnostic
laboratory, culture of Cryptococcus is not a significant health
hazard, because only the yeast form is grown routinely, and
this form does not aerosolize from media. During handling
of infected tissues or cultures, precautions should be taken to
prevent inadvertent cutaneous inoculation of the organism.
Veterinarians should consider patient sedation for procedures
that involve fine-needle aspiration in order to avoid inadver-
tent needle-stick injuries, which have caused localized cuta-
neous infection in the human health care setting.63 Because
sporotrichosis is a major differential diagnosis for cats with
cutaneous lesions, gloves should always be worn and hand
hygiene performed when handling cats with suspicious lesions
(see Chapter 64).
acid and enrofloxacin for 7 days), and he was treated with
subcutaneous fluids (lactated Ringer’s solution, 150 mL,
once). Over the following 5 days his clinical signs lessened,
but then anorexia developed. Physical examination 2 days
after the onset of anorexia revealed a rectal temperature
of 102.5°F (39.2°C) and weight loss (3.7 kg at the previous
visit to 3.2 kg). A CBC and biochemistry panel showed mild
lymphopenia (1157 cells/µL, reference range 1500-7000
cells/µL). Treatment with metronidazole (39 mg/kg PO q24h)
and prednisone (3 mg/kg PO q24h) was instituted. Three
days before he was brought to the VMTH, he began panting
and vocalizing. The metronidazole was discontinued and the
prednisone dose was reduced to 1.5 mg/kg PO q24h. The
following day, the owners noted that Simon’s eyelids were
twitching, and he urinated and defecated on himself in the
litter box. Clindamycin (6 mg/kg PO q12h) was prescribed
Continued
610 SECTION 3  Fungal and Algal Diseases
for suspected toxoplasmosis, and Simon was referred for
further evaluation.
Simon had no other previous medical or surgical history
apart from being neutered as a kitten. He had access to
the outdoors and there were no other cats in the house-
hold. There had been no changes in his thirst or urina-
tion, no vomiting or diarrhea, and he was last vaccinated
at 5 years of age. He had killed a large rat ­approximately
5 weeks earlier and had not been himself since.
Physical Examination:
Body Weight: 2.9 kg.
General: Obtunded but responsive, estimated as 5%
dehydrated. T = 100.2°F (37.9°C), HR = 200 beats/min, RR =
60 breaths/min, CRT <2 s.
Eyes, Ears, Nose, and Throat: Bilateral mydriasis and
absent pupillary light reflexes were noted. There was no
evidence of nasal discharge and nasal airflow was normal.
Mucous membranes were pink and slightly tacky. Fundic
examination was within normal limits.
Musculoskeletal: Body condition score 4/9, ambulatory. The
cat had a slow gait with a short stride.
Respiratory: Tachypnea was present with a normal respiratory
effort. Normal breath sounds were present on auscultation.
Cardiovascular, Gastrointestinal, Genitourinary and
Lymph Nodes: No clinically significant abnormalities were
detected. The urinary bladder was small.
Neurologic Examination: The cat was mildly obtunded,
walked with a crouched stance and behaved as if nonvisual.
Bilateral mydriasis and absent menace and pupillary light
reflexes were confirmed. Intermittent horizontal nystagmus
with the fast phase to the left was present. Cranial nerve
examination was otherwise normal. Placing and hopping
reactions were delayed in the left pelvic limb. There was a
decreased left patellar reflex, but other segmental reflexes
were intact. There was no apparent pain on spinal palpation,
but the cat appeared painful when the tail was raised.
Neuroanatomic location: Multifocal CNS disease.
Laboratory Findings:
CBC:
HCT 27.2% (30%-50%)
MCV 48.3 fL (42-53 fL)
MCHC 29.8 g/dL (30-33.5 g/dL)
Reticulocytes 8400 cells/µL (7000-60,000 cells/µL)
WBC 12,360 cells/µL (4500-14,000 cells/µL)
Neutrophils 11,000 cells/µL (2000-9000 cells/µL)
Lymphocytes 828 cells/µL (1000-7000 cells/µL)
Monocytes 445 cells/µL (50-600 cells/µL)
Eosinophils 87 cells/µL (150-1100 cells/µL)
Basophils 0 cells/µL (0-50 cells/µL)
Platelets 357,000/µL (180,000-500,000 platelets/µL).
Serum Chemistry Profile:
Sodium 150 mmol/L (151-158 mmol/L)
Potassium 4.3 mmol/L (3.6-4.9 mmol/L)
Chloride 115 mmol/L (117-126 mmol/L)
Bicarbonate 19 mmol/L (15-21 mmol/L)
Phosphorus 5.4 mg/dL (3.2-6.3 mg/dL)
Calcium 10 mg/dL (9.0-10.9 mg/dl)
BUN 21 mg/dL (18-33 mg/dL)
Creatinine 1.2 mg/dL (1.1-2.2 mg/dL)
Glucose 136 mg/dL (63-118 mg/dL)
Total protein 7.5 g/dL (6.6-8.4 g/dL)
Albumin 3.8 g/dL (2.2-4.6 g/dL)
Globulin 3.7 g/dL (2.8-5.4 g/dL)
ALT 37 U/L (27-101 U/L)
AST 28 U/L (17-58 U/L)
ALP 8 U/L (14-71 U/L)
GGT < 3 U/L (0-4 U/L)
Cholesterol 177 mg/dL (89-258 mg/dL)
Total bilirubin < 0.1 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.018; pH 7.0; 25 mg/dL protein; no bilirubin,
hemoprotein, glucose; rare WBC/HPF, 0 RBC/HPF, few
amorphous crystals, few lipid droplets.
ELISA for FeLV Antigen and FIV Antibody: Negative.
Latex Agglutination for Cryptococcus Antigen: Positive at
1:32,768.
Imaging Findings:
Plain Thoracic and Abdominal Radiographs: The only
abnormality present was mild lateral spondylosis of the
caudal lumbar spine.
Abdominal Ultrasonography: The liver was mildly enlarged
and contained multiple small hyperechoic foci. In the
kidneys, mild cortical hyperechogenicity and decreased
corticomedullary distinction were present.
Magnetic Resonance Imaging (Brain): Precontrast images
showed a well-defined, round T2-hyperintense, T1-
hypointense lesion in the gray matter of the right cerebral
cortex at the junction of the parietal and occipital lobes
(see Figure 62-7). Postcontrast images showed moderate
contrast enhancement of this lesion. There was mild contrast
enhancement of the meninges ventral to the right preoptic
area. There was moderate, focal contrast enhancement of
the gray matter at the lateral aspect of the right pyriform
lobe. There was contrast enhancement of the medial
aspect of both pyriform lobes adjacent to the hypophysis.
Impressions: Multifocal contrast-enhancing lesions with T2
hyperintensity and a cystic lesion in the right cerebral cortex.
Cytologic Findings: CSF analysis (cisternal): Protein
concentration 29 mg/dL (reference range, <25 mg/dL). The
specimen had a moderate increase in cellularity with a low
number of erythrocytes. Numerous thick-capsuled yeast
organisms with frequent narrow budding forms were noted,
consistent with Cryptococcus spp. Nucleated cells consisted
of nondegenerate neutrophils admixed with low numbers
of variably vacuolated macrophages and small lymphocytes.
The differential cell count was 83% neutrophils, 7% small
mononuclear cells, and 10% large mononuclear cells.
Microbiologic Testing: Fungal culture (CSF): Cryptococcus
gattii. Multilocus sequence typing revealed the organism
belonged to C. gattii molecular type VGIIa.
Diagnosis: Cryptococcus gattii meningoencephalitis.
Treatment: Simon was treated with deoxycholate
amphotericin B (0.25 mg/kg in 30 mL 5% dextrose IV over
30 minutes on Monday, Wednesday, and Friday for five
treatments). Before and after this treatment, 0.9% NaCl
was administered at twice maintenance rate and kidney
analytes were monitored. Flucytosine (62.5 mg PO q8h)
and prednisone (2.5 mg PO q24h) were also administered.
An esophagostomy tube was placed for enteral nutrition
because of persistent inappetence. By day 7, Simon
appeared more alert, pupillary light reflexes and menace

responses were present but weak, and nystagmus had
resolved. He was discharged from the hospital on day 12,
with instructions to continue treatment with fluconazole
(50 mg PO q12h) and prednisone (2.5 mg PO q48h), in
addition to esophagostomy tube feeding. On day 24, he
was reevaluated for lethargy of several days’ duration.
Physical examination showed resolution of cranial nerve
abnormalities, but delayed hopping reactions in all
limbs were present. He was treated with six additional
amphotericin B treatments over 2 weeks. He began
eating, and the esophageal feeding tube was removed
on day 40. Three months after diagnosis, his neurologic
signs had almost completely resolved with the exception
of occasional tremors, persistent mild placing reaction
deficits, and lumbosacral pain. A cryptococcal antigen
titer was positive at 1:65,536. The fluconazole was replaced
with itraconazole solution (3 mg/kg PO q12h). After an
SUGGESTED READINGS
Datta K, Bartlett KH, Baer R, et al. Spread of Cryptococcus gattii into
Pacific Northwest region of the United States. Emerg Infect Dis.
2009;15:1185-1191.
Kronstad JW, Attarian R, Cadieux B, et  al. Expanding fungal patho-
genesis: Cryptococcus breaks out of the opportunistic box. Nat Rev
Microbiol. 2011;9:193-203.
Trivedi SR, Malik R, Meyer W, et  al. Feline cryptococcosis: impact
of current research on clinical management. J Feline Med Surg.
2011;13:163-172.
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additional month, the antigen titer was 1:1024, a serum
chemistry profile was unremarkable, and the owners felt
that Simon was a normal cat. The prednisone treatment
was discontinued thereafter. A year later, the antigen titer
was 1:20. At a recheck examination 2 years later the cat was
neurologically normal. The owner declined further antigen
testing and chose to continue treatment with itraconazole.
Comments: This cat had CNS cryptococcosis caused by
Cryptococcus gattii molecular type VGIIa, a hypervirulent
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CHAPTER 63
Coccidioidomycosis
Jane E. Sykes
Overview of Coccidioidomycosis
First Described: 1892 (by the medical student Alejandro Posa-
das), in a person from Buenos Aires, Argentina, where it
was initially mistaken for a protozoan (Coccidia)1
Cause: Coccidioides immitis and Coccidioides posadasii (asco-
mycetes, no known teleomorph)
Affected Hosts: Primarily humans and dogs, but also a variety
of other animals that include cats, domestic farm animal
species, and a variety of wildlife hosts
Geographic Distribution: Southwestern United States, Mex-
ico, and parts of Central and South America (northeastern
Brazil, Argentina, Paraguay, and Bolivia)
Mode of Transmission: Inhalation of arthroconidia from the
environment; rarely cutaneous inoculation
Major Clinical Signs: Cough, fever, inappetence, weight loss,
tachypnea, increased respiratory effort, lameness, subcu-
taneous masses or draining skin lesions, lymphadenopa-
thy, neurologic signs, ascites due to right-sided heart
failure (pericarditis), ocular lesions (uveitis, chorioretinitis,
endophthalmitis)
Differential Diagnoses: Neoplasia, other deep mycoses, myco-
bacteriosis, actinomycosis or nocardiosis, toxoplasmosis
or neosporosis, systemic immune-mediated disease
Human Health Significance: Direct transmission from ani-
mals to humans does not usually occur, although sharps
injuries or bite wounds from affected animals may be a
source of human infection. Dogs are a sentinel for human
exposure.
Etiology and Epidemiology
Coccidioides spp. are dimorphic, soil-borne fungi that exist
in the environment as a mycelium and in tissues (at 37°C) as
peculiar structures known as spherules. The mycelium consists
of chains of haploid, multinucleated, barrel-shaped arthroco-
nidia (or arthrospores) that alternate with smaller, thin-walled,
nonviable cells. The arthroconidia remain viable in the soil for
long periods. They ultimately fragment from each other and are
subsequently aerosolized and inhaled by animal hosts (Figure
63-1). This can be followed by the development of coccidioi-
domycosis, a localized pulmonary disease or a serious multisys-
temic disease if dissemination occurs in the face of inadequate
immune responses.
In the environment, Coccidioides spp. are distributed in
the lower Sonoran life zone, regions characterized by semiarid
to arid soils, low elevations above sea level, and hot summers.
Worldwide, Coccidioides spp. are found in the southwest-
ern United States, Mexico, and parts of Central and South
America, specifically Colombia, Guatemala, Honduras, Ven-
ezuela, northeastern Brazil, Argentina, Paraguay, and Bolivia
(Figure 63-2). In the southwestern United States, highly
endemic regions are the south-central valley of California,
especially Bakersfield (“valley fever”) and Arizona, especially
Tucson and Phoenix.2 Infections also occur in non-endemic
regions in dogs and humans that have a travel history to
endemic regions.
Two species of Coccidioides have been identified, C. posa-
dasii and C. immitis. These appear to cause similar clinical
manifestations and have similar antifungal drug suscepti-
bilities, but further studies are required.3 The geographic
range of C. immitis is largely limited to the central valley of
California, whereas C. posadasii is found elsewhere.4 How-
ever, there may be some overlap in the distribution of these
organisms.
Infection of animals and humans with Coccidioides spp.
often follows a cycle of moist conditions (required for growth
of the organism in the soil), a dry period, then soil disrup-
tion, such as may occur with heavy rainfall, earthquakes, dust
storms, prolonged droughts, or construction.5-7 Dogs of any
breed, age, or sex may be affected, although large-breed, young
adult dogs predominate in case series.8,9 In dogs seen at the
University of California Veterinary Medical Teaching Hospi-
tal, boxers, poodles, pointers, Labrador and golden retrievers,
German shepherds, Australian shepherds, Dalmatians, Scottish
terriers, dachshunds, and beagles are overrepresented when
compared with the hospital patient population.10 Risk factors
for infection in dogs from Arizona include being housed out-
doors during the day rather than indoors, roaming areas more
than 1 acre, and walking in the desert.11 Latent infections may
also occur in dogs, and these can reactivate after treatment
with immunosuppressive drugs, such as corticosteroids and
chemotherapeutic agents, including after previous recovery
from coccidioidomycosis.
Although not as prevalent as in dogs, coccidioidomycosis
also occurs in cats.12 In a study of 48 cats from Arizona, cats
ranged in age from 1 to 15 years (median, 5 years) and there was
no obvious sex or breed predisposition.12 One cat was infected
with FIV, and all cats tested were FeLV negative. Interestingly,
a search of the author’s hospital and pathology database from
1990 to 2012 revealed not a single cat with coccidioidomycosis,
which might suggest cats are less susceptible to C. immitis infec-
tion than to C. posadasii infection.
613
614 SECTION 3  Fungal and Algal Diseases

FIGURE 63-1  Life cycle of Coccidioides spp. Hyphae fragment into arthroconidia, which are inhaled and form spherules in tissues. The spherules rupture and release endospores, which
form new spherules. In some dogs, the organism disseminates from the lungs to a variety of other tissues, but especially local lymph nodes, bone, skin, the pericardium, the eye, and the
central nervous system.

Clinical Features
Signs and Their Pathogenesis
The clinical signs of coccidioidomycosis depend on factors
such as the infectious dose and the immune status of the host.
Arthroconidia are inhaled and are phagocytosed by alveolar
macrophages. They then enlarge into a spherule, which mea-
sures 8 to 100 µm in diameter. Hundreds of endospores then
develop within the spherule, which are released when the mature
spherule ruptures. These attract neutrophils, which leads to a
pyogranulomatous inflammatory response. Each released endo-
spore that escapes immune destruction then enlarges into a
new spherule, and the cycle continues. Endospores can revert
to mycelial growth when removed from the site of infection. In
hosts that are unable to mount an effective immune response,
endospores disseminate by lymphatics to tracheobronchial
lymph nodes and hematogenously to a variety of body sites.
Inoculation coccidioidomycosis can rarely occur, and generally
results in lesions that are localized to the site of the wound. In
one report, it followed removal of a foxtail from a wound.13
Most infections in endemic areas are subclinical.14 Develop-
ment of clinical signs often follows a subacute to chronic course,
Highly endemic
with variable and often mild systemic signs such as intermittent
Moderately endemic
fever, lethargy, inappetence, and weight loss.8,15 Many dogs oth-
Mildly endemic
erwise appear healthy, with occasional periods of inappetence.8
Suspected endemic
Clinical signs may be present for days to years before veteri-
nary attention is sought.9 Signs of respiratory tract involvement
include a harsh cough, increased respiratory effort, tachypnea
and/or exercise intolerance.9,15 Cough may be chronic and is
FIGURE 63-2  Geographic distribution of Coccidioides spp. worldwide. The inset
sometimes associated with gagging or retching; occasionally it
shows the distribution of Coccidioides species in the southwestern United States.
results from bronchial compression by enlarged tracheobron-
chial lymph nodes. Rarely, diffuse pneumonia follows a fulmi-
nant course, with signs that resemble acute bacterial pneumonia
or septic shock, as reported in immunocompromised human
patients.2

FIGURE 63-3  Draining skin lesion on the left elbow of a 5-year-old shepherd mix
with disseminated coccidioidomycosis. Radiography revealed a mixed productive and
destructive lesion of the distal humerus and proximal ulna. Productive lesions of the sixth
and seventh ribs were also present. Arthrocentesis revealed suppurative inflammation
with no growth on culture. Diagnosis was ultimately made by biopsy of the synovium and
serology. The Coccidioides titer was 1:64.
A smaller percentage of infected dogs develop disseminated
infections. Sites of dissemination often include osteoarticular
sites, the central nervous system (CNS), skin, peripheral lymph
nodes, eyes, testes, prostate, and the pericardium.9,10 Rarely,
sites such as the liver, spleen, gastrointestinal tract, or urinary
system are affected.8 Sometimes dissemination to only one ana-
tomic site is evident; in other dogs multiple sites are involved. A
history of respiratory signs may be absent.8 Bone involvement
may manifest as lameness and/or one or more firm swellings
associated with the appendicular or axial skeleton. Dogs with
skin involvement may have subcutaneous masses ulcerated
skin lesions that drain serosanguineous fluid (Figure 63-3).8
Skin lesions may overlie sites of osteomyelitis. Neurologic signs
result from meningoencephalitis and include obtundation,
blindness, nystagmus, absent menace reflexes, diminished gag
responses, ataxia, abnormal placing reactions, pacing, circling,
cervical pain, tetraparesis, and seizures.9,16,17 Ocular mani-
festations of coccidioidomycosis include chorioretinitis, uve-
itis, optic neuritis, and endophthalmitis.9,18,19 Involvement of
abdominal organs such as the liver and gastrointestinal tract
can lead to inappetence, vomiting, and/or diarrhea.8 Pericardi-
tis may lead to signs of right-sided cardiac failure, with ascites
and pleural effusion.8
Physical Examination Findings
Fever is present in up to two thirds of dogs with coccidioidomy-
cosis (range, 103°F to 106°F) on physical examination. Other
common findings in dogs are lethargy, weakness, thin body
condition or profound muscle atrophy, tachypnea, increased
respiratory effort, inducible or spontaneous cough, and/or
increased lung sounds.9 Lameness and firm, often painful bony
swellings with associated muscle atrophy may be present in
dogs with osteomyelitis. Less often, focal or (very rarely) gen-
eralized peripheral lymphadenomegaly, subcutaneous masses or
draining skin lesions, or swollen joints are present. Dogs with
CHAPTER 63  Coccidioidomycosis 615
pericardial coccidioidomycosis may have abdominal distention
with a palpable fluid wave, tachycardia, tachypnea, jugular
pulses, muffled breath and heart sounds due to pleural effu-
sion, mucosal pallor, prolonged capillary refill time, and weak
pulses.20 Neurologic signs, when present, include obtundation,
apparent blindness, ataxia, nystagmus, spinal pain, circling,
and tetraparesis. The most common ocular abnormality is uve-
itis, but keratitis, conjunctivitis, granulomatous chorioretinitis,
optic neuritis, retinal detachment, and endophthalmitis with
secondary glaucoma have also been described.9,18,19
Of 48 cats with coccidioidomycosis, fever was present in
31%. Skin lesions were present in more than 50% of 48 affected
cats and included draining skin lesions, subcutaneous granulo-
mas, and abscesses.12 Tachypnea or increased respiratory effort
was present in 25% of affected cats, and lameness in fewer than
20% of cats. Ocular abnormalities were present in 13% of cats.
Conjunctival masses, periorbital swellings, chorioretinitis, reti-
nal detachment, endophthalmitis, and/or anterior uveitis have
been described.21,22 A small percentage of cats have neurologic
signs such as hyperesthesia, posterior paresis, seizures, and
ataxia. Dissemination to abdominal organs such as the spleen
can occur.23
Diagnosis
Coccidioidomycosis is usually suspected based on a history of
travel to or residence in an endemic region, and consistent clini-
cal signs and radiographic findings. Because the disease is not
prevalent in some endemic regions (such as coastal southern
California), early diagnosis requires a high index of suspicion
in these regions. The diagnosis can be confirmed with cytologic
examination of aspirates or body fluids, serologic assays that
detect antibodies to Coccidioides spp., histopathology, and/or
fungal culture (Table 63-1).
Laboratory Abnormalities
Complete Blood Count
CBC findings in dogs with coccidioidomycosis are nonspecific
and resemble those of other deep mycoses. Most affected dogs
have mild, normocytic, normochromic, nonregenerative anemia
and mild leukocytosis. Leukocytosis results from a neutrophilia,
occasionally with a left shift and evidence of neutrophil toxic-
ity.9,20 A monocytosis and lymphopenia may also be present,
but in contrast to human infections, eosinophilia is not typically
present.
Serum Biochemical Tests
Almost all dogs with coccidioidomycosis have hypoalbumin-
emia, and approximately half of affected dogs and cats have
hyperglobulinemia (up to 7.8 mg/dL in one study).9,12 Hypoal-
buminemia is usually mild to moderate, but rarely may be more
severe (<1.5 g/dL). Uncommonly, mild hypercalcemia is present.
Increased activity of serum ALP may be present in some dogs,
possibly as a result of bone involvement.
Urinalysis
The urinalysis is often normal, but significant proteinuria (urine
protein-to-creatinine ratios as high as 9.5, reference range <1)
has been reported in some dogs with coccidioidomycosis.9 It
is not clear whether proteinuria of this magnitude results from
glomerulonephritis secondary to chronic immune stimulation or
another comorbidity.
616 SECTION 3  Fungal and Algal Diseases

TABLE 63-1
Diagnostic Assays Currently Available for Coccidioidomycosis in Dogs and Cats
Assay Specimen Type Target Performance
Cytologic Aspirates of affected tissues, Coccidioides spherules Low sensitivity and organisms may be difficult to
examination body fluids, impression and/or endospores accurately identify in some specimens. Immature
smears of skin lesions spherules may be mistaken for other fungi or cel-
lular aggregates or debris.
Fungal culture Aspirates or biopsies of affect- Coccidioides spp. Generally sensitive and specific, but false negatives
ed tissues, body fluids such are possible if specimen size is low. Can require
as CSF or pleural effusion, several weeks’ incubation. A significant labora-
transtracheal or bronchoal- tory health hazard.
veolar lavage specimens
Antibody serology Serum, CSF IgG or IgM antibodies Sensitivity is close to 100%. Quantification of an
(gel immunodiffu- to Coccidioides spp. IgG antibody titer is possible. Titers decline with
sion) successful treatment. In hyperendemic areas,
titers as high as 1:16 have the potential to exist
in subclinically infected or recovered dogs, so
positive serology should be supported with other
diagnostic tests for coccidioidomycosis.
Serology for Serum, urine Coccidioides spp. Low sensitivity (≤20%); false positives can occur in
Coccidioides galactomannan antigen dogs with histoplasmosis or blastomycosis. Not
antigen recommended.
Real-time PCR Biopsies, body fluids, respi- Coccidioides DNA Not yet validated in adequate numbers of dogs or
assays ratory lavage specimens, cats with coccidioidomycosis; usefulness requires
aspirates further evaluation.
Histopathology Biopsy specimens from Coccidioides spp. Limited sensitivity in dogs; submission of multiple
affected tissues, such spherules biopsies is recommended when possible. Often
as bone biopsies only one or two spherules are visualized. Special
stains such as periodic acid–Schiff and silver
stains facilitate organism detection.

Cerebrospinal Fluid Analysis
CSF analysis in dogs with CNS coccidioidomycosis usually reveals
an increase in protein concentration (generally <300 mg/dL,
reference range <25 mg/dL) and a mild to moderate increase in
the nucleated cell count (usually <50 cells/µL, reference range
<2 cells/µL).9 The differential cell count may show mixed, pre-
dominantly lymphocytic, mixed mononuclear, or neutrophilic
pleocytosis. Organisms are generally not observed.
Diagnostic Imaging
Plain Radiography
Thoracic radiographs in dogs with disseminated coccidioi-
domycosis are often unremarkable. The most common radio-
graphic finding in dogs with pulmonary coccidioidomycosis
is mild to severe hilar lymphadenomegaly (Figure 63-4, A),
which was present in 10 of 19 dogs with coccidioidomycosis
from California.9 Hilar lymphadenopathy can also be present
in affected cats.12 Although hilar lymphadenopathy can be the
only finding, most dogs with hilar lymphadenopathy also have
pulmonary infiltrates, which are usually mild to moderate and
interstitial. Nodular interstitial, interstitial-alveolar, bronchoin-
terstitial infiltrates, and/or sternal lymphadenomegaly can also
be present (Figure 63-4, B and C).9,15 Dogs with pericarditis can
have cardiomegaly and pleural effusion, with hepatomegaly and
decreased abdominal detail.20
Radiographs of affected bones in both dogs and cats with
Coccidioides osteomyelitis reveal local periosteal proliferation
as well as osteolysis and soft tissue swelling. Lesions can resem-
ble osteosarcomas (Figure 63-5).8,12
Sonographic Findings
Abdominal ultrasound examination of dogs with coccidioido-
mycosis is typically unremarkable. Mild hepatosplenomegaly
and abdominal lymphadenomegaly have been described in
a small proportion of dogs.9 Ascites, hepatomegaly, and dis-
tended hepatic veins may be evident in dogs with pericarditis.20
Echocardiography in dogs with Coccidioides spp. pericardi-
tis can reveal thickening and mass lesions of the pericardium,
adherence of the pericardium to the epicardium, pericardial
and pleural effusion, and evidence of cardiac tamponade (see
Figure 86-3).20
Advanced Imaging
Advanced imaging findings in dogs with coccidioidomyco-
sis have not been extensively reported in the literature. Focal,
contrast-enhancing intra-parenchymal mass lesions in the brain
or osteomyelitis of the skull may be identified on computed
tomography (CT) of the head.24 Thoracic CT findings include
contrast-enhancing miliary nodular pulmonary lesions; single or
multifocal larger nodules or masses; tracheobronchial, sternal,

A or a reactive lymphocyte population. However, occasionally a
B sidered when cytology of tissue aspirates, body fluids, or lung
C ties of canine serum.25 The complement fixation test is positive
FIGURE 63-4  Thoracic radiographic patterns in coccidioidomycosis. A, Lateral thoracic
radiograph from a 7-year-old intact male English pointer that shows several tracheobronchial
lymphadenomegaly and focal pulmonary infiltrates. B, Lateral thoracic radiograph from a
3-year-old intact female Rottweiler with cough, anorexia, and respiratory distress. There
are diffuse nodular and peribronchial infiltrates throughout the pulmonary parenchyma
and possible hilar lymphadenopathy. Necropsy revealed necrogranulomatous interstitial
pneumonia with intralesional spherules. The spleen, liver, and kidney were also involved. 
C, Dorsoventral thoracic radiograph from a 2-year-old female spayed Labrador retriever with
cough and inappetence. There is an alveolar pattern in the left cranial lung lobe.
CHAPTER 63  Coccidioidomycosis 617
or mediastinal lymphadenopathy; focal regions of alveolar or
peribronchial infiltrates; lobar consolidation; pleural or medi-
astinal thickening; and, rarely, pleural effusion (Figure 63-6).
MRI of the brain in dogs with CNS coccidioidomyco-
sis may show focal hyperintense lesions within the brain on
T2-weighted images (Figure 63-7). These are typically isointense
on T1-weighted images and enhance with contrast. Other find-
ings include ventricular dilation and/or meningeal enhancement.
Microbiologic Tests
Cytologic Examination
Cytologic examination of aspirates of affected tissues, pleu-
ral effusion, or respiratory lavage specimens typically reveals
granulomatous or pyogranulomatous inflammation, sometimes
with rare multinucleated giant cells, scattered eosinophils, and/
suppurative inflammatory response predominates. Spherules
may be visualized as round, deeply basophilic, double-walled,
slightly crinkled structures that range in diameter from 8 to
70 µm. Spherules are generally low in number, frequently asso-
ciated with cellular aggregates, but often they are not evident
or easily identified. Usually their internal structure is not easily
discernible, but occasionally endospores are seen in association
with ruptured spherules (Figure 63-8). The endospores are 2 to 5
µm in diameter, are surrounded by a thin, nonstaining halo, and
have small, round to oval, densely aggregated, eccentric nuclei.
Endospores may be found extracellularly or within phagocytes.
In one report, only endospores were visualized in a fine-needle
aspirate from a bone lesion; intact spherules were not seen.13
Because cytology is relatively insensitive for diagnosis of
coccidioidomycosis when compared with other deep mycoses,
serologic testing for coccidioidomycosis should always be con-
washes reveals pyogranulomatous, suppurative, or granuloma-
tous inflammation but organisms are not seen, and exposure to
Coccidioides is possible based on the history.
Serologic Diagnosis
Serologic assays that detect antibodies to Coccidioides spp.
are important diagnostic tests for coccidioidomycosis because
organism detection assays have low sensitivity. Most diagnostic
laboratories screen serum specimens with qualitative gel immu-
nodiffusion (ID) assays for IgG or IgM antibodies (see Chapter
2). Detection of IgM (tube precipitin) or IgG (complement fixat-
ing) antibodies is reported, depending on the antigen prepara-
tion used in the assay. In dogs, IgM is detectable within 2 to
5 weeks of infection, and IgG appears after 8 to 12 weeks.10
Quantification of an IgG antibody response can then be per-
formed by quantitative ID, whereby serial dilutions of patient
serum are subjected to gel ID.14 In human patients, the com-
plement fixation test is often used to quantify IgG antibody,
but frequent false-positive results have been described with
this method in dogs as a result of anticomplementary proper-
in most cats with coccidioidomycosis; titers have ranged from
1:2 to 1:128.12
False-negative serologic test results are extremely rare in dogs
with coccidioidomycosis tested at the author’s institution,9,20
but they have been described in dogs from Arizona.26,27 Anti-
body titers usually range from 1:2 to 1:256; the median titer
was 1:32 in one study.9 There is no clear correlation between
the magnitude of the IgG titer and the severity of illness in dogs.
618 SECTION 3  Fungal and Algal Diseases

A B C
FIGURE 63-5  Osteomyelitis caused by Coccidioides spp. A, Left tarsus of a 4-year-old female spayed Dalmatian that was evaluated for signs of right-sided congestive heart failure due
to pericarditis. Severe osteolysis is present. B, Right humerus of a 4-year-old female spayed shepherd mix. There is a mixed productive and lytic lesion of the distal diaphysis of the right
humerus. C, Lesion in (B) at necropsy. (Courtesy University of California, Davis, Veterinary Anatomic Pathology Service.)

FIGURE 63-6  Computed tomography scan of the thorax of a 2-year-old intact male
Labrador retriever with coccidioidomycosis. There are infiltrates in the caudal aspect of the
right cranial lung lobe that extend from the mediastinum to the pleural surface. The walls
of the right cranial bronchus and associated airways are thickened. Bronchial lymphadeno-
megaly was also identified.
B
FIGURE 63-7  MRI scan of the brain of a 7-year-old male neutered shih tzu with
disseminated coccidioidomycosis. A, A small focus of increased signal intensity on
T2-weighting is present in the left lateral aspect of the brainstem (arrowhead). Ventricular
dilation is also present (arrow). B, T1-weighted image after administration of gadolinium.
The lesion in the brainstem is contrast enhancing.

A Real-time PCR assays have been used for rapid detection of
B lar bony swellings with associated draining skin lesions, and/or
FIGURE 63-8  Cytologic appearance of Coccidioides spp. spherules. A, Spherules often
appear as deeply basophilic, crinkled, intact, or ruptured ball-like structures (white arrow).
B, Sometimes, a refractile spherule wall is appreciated (arrow) and endospores are visual-
ized as granular material within the spherule.
Dogs with active infection can have titers as low as 1:2, and
healthy dogs in hyperendemic areas in Arizona can have titers
as high as 1:16.14 As a result, positive titers of 1:16 or lower
must be interpreted in light of clinicopathologic abnormalities,
and in hyperendemic areas, other diagnostic tests (such as
­culture, cytology or histopathology) should be used to ­confirm
the diagnosis if possible. Latex agglutination and ELISA (EIA)
assays for detection of anti-Coccidioides antibody have also
been developed, but these are not widely used in veterinary
medicine, and their use has been associated with a significant
number of false-positive reactions in humans.2,25,28
A serologic assay that detects Coccidioides antigen in urine is
available for diagnosis of coccidioidomycosis in human patients,
such as in immunocompromised patients who lack the ability to
produce significant quantities of antibody. However, preliminary
CHAPTER 63  Coccidioidomycosis 619
data suggest that this assay has unacceptable sensitivity (≤20%)
for diagnosis of coccidioidomycosis in dogs when serum, urine, or
both specimens are tested, and false-positive reactions occur in a
small proportion of dogs with blastomycosis or histoplasmosis.29
Fungal Culture
Coccidioides spp. can be isolated from clinical specimens on rou-
tine fungal media in the laboratory, but a negative culture result
does not rule out coccidioidomycosis. Growth of a gray-white,
downy mold that is tan to brown when visualized through the
bottom of the plate usually appears after incubation for several
days. Isolates can be identified based on their light microscopic
appearance, with or without molecular methods. Culture of
Coccidioides spp. is a serious laboratory health hazard and
should only be performed if necessary and by suitably equipped
laboratories. The clinician should warn the laboratory of the
possibility of dimorphic fungal infection if suspected.
Molecular Diagnosis Using the Polymerase Chain Reaction
Coccidioides spp. in respiratory specimens or CSF from affected
humans, although sensitivity has been limited.2,30 PCR assays
have also been used in epidemiologic studies to identify major
growth sites of Coccidioides spp. in soil.31 PCR assays have not
yet been widely applied to the diagnosis of coccidioidomycosis
in dogs and cats.
Pathologic Findings
Gross pathologic findings in dogs with coccidioidomycosis
include mottling and consolidation of the pulmonary paren-
chyma; single or multiple pale masses, nodular lesions, or
emphysematous bullae within the lungs (Figure 63-9); enlarged
and firm tracheobronchial, mediastinal, or sternal lymph nodes;
plaque-like lesions on the pleura; and pleural and pericardial
effusion. The interior of masses or nodules may be firm, caseous,
or liquefied. The pericardium and epicardium may be thickened
with mass lesions and adhesions between the pericardium and
epicardium.20 Ascites may also be present in dogs with pericar-
ditis. Dogs with disseminated disease may have diffuse muscle
atrophy, one or more enlarged peripheral lymph nodes, irregu-
evidence of arthritis and synovitis. Meningoencephalitis may be
evident as mass lesions within the brain parenchyma, ventricu-
lar dilation secondary to obstruction of CSF flow by inflam-
matory lesions, regional discoloration within the brain, and/
or discoloration and thickening of the meninges. Rarely, pros-
tatic enlargement, hepatic congestion and friability, multifocal
nodular lesions within the myocardium or other parenchymal
organs, and ocular lesions are present (see Physical Examination
Findings for a description of ocular lesions).
Histopathology of affected tissues reveals large numbers of
neutrophils and macrophages and sometimes marked fibrosis,
with occasional endosporulating or non-endosporulating spher-
ules. Multinucleated giant cells may be present and can contain
spherules.26 Spherules have a thick wall, and endospores may
be visualized in mature spherules (Figure 63-10). Often only
one or two spherules are visualized, and in some cases they are
not found at all. Use of special stains such as periodic acid–
Schiff and Gomori’s methenamine silver can enhance organism
identification within lesions. The presence of large numbers of
spherules has been described in some affected cats.23,32 Chronic,
diffuse glomerulonephritis was present in 3 of 13 dogs with
620 SECTION 3  Fungal and Algal Diseases
FIGURE 63-9  Lungs of the dog in Figure 63-4, B. Multiple pale nodules are present throughout the lung parenchyma.
FIGURE 63-10  Histopathology of the tarsus of the dog in Figure 63-5, A. A single
mature spherule is surrounded by a pyogranulomatous inflammatory response. The spher-
ule contains many endospores.
coccidioidomycosis that had full necropsy examinations at the
author’s hospital; an additional dog with renal lesions had mul-
tiple renal pyogranulomas with intralesional spherules.24
Treatment and Prognosis
Successful treatment of coccidioidomycosis is limited by the high
cost of treatment and the often advanced nature of the disease
by the time it is diagnosed in some dogs. Treatment consists of a
combination of antifungal drug therapy and supportive care; in
some cases, surgery is required. Usually a minimum of 6 months
of treatment is required.
Antimicrobial Treatment
Animals with coccidioidomycosis can respond well to azole
monotherapy with fluconazole or itraconazole. Ketoconazole
has been used successfully to treat dogs and cats in the past but
is no longer the drug of first choice owing to its lower activity
and increased rate of adverse effects. Fluconazole is the least
expensive azole and may be a better choice than itraconazole
for dogs with CNS involvement, because of its superior penetra-
tion of the CNS. The dose of fluconazole used by the author is
5 mg/kg PO q12h. Itraconazole (5 mg/kg PO q24h) is preferred
for animals with bone involvement and may be effective in dogs
that fail to respond to treatment with fluconazole. In human
patients, itraconazole demonstrated a greater response rate in
a blinded comparison with fluconazole.33 Compounded formu-
lations of itraconazole should be avoided. Small dogs are best
treated with itraconazole solution (10 mg/mL), which has higher
bioavailability than the capsules (see Chapter 9). If capsules are
used, they should be administered with food. If clinical signs do
not improve with azole treatment, therapeutic drug monitoring
could be considered to ensure that serum drug concentrations
are adequate. Periodic monitoring of liver enzymes is recom-
mended for animals treated with azole antifungals.
Treatment with deoxycholate or lipid-complexed amphoteri-
cin B is recommended either alone or in combination with an
azole for dogs with refractory or severe disease (see Chapter 9
for dosing protocols). However, studies that compare ampho-
tericin B to azole monotherapy in dogs have not been performed.
Voriconazole and posaconazole have been used to treat refrac-
tory coccidioidomycosis in human patients, but they are not con-
sidered first-line drugs.2 Their use for treatment of affected dogs
or cats is limited by expense. Voriconazole could be considered
for treatment of refractory meningoencephalitis if client finances
permit its use, because it has excellent penetration of the CNS.
Surgical Management
Ultimately, some dogs with severe and persistent osteomyelitis
require amputation to control the infection, and enucleation
may be required for dogs with endophthalmitis. Successful treat-
ment of Coccidioides pericarditis has been reported after sub-
total pericardiectomy and epicardial excision, which should be
performed in conjunction with antifungal drug therapy. Refer-
ral to a specialist surgeon with experience with the technique is
strongly recommended. The perioperative mortality rate in one
study was reported as 24%, and the 2-year survival rate after
discharge was 82%.20
Supportive Care
Other treatments that may be required for dogs with severe
pulmonary coccidioidomycosis are similar to those described

for blastomycosis and include oxygen supplementation, nebu-
lization and coupage, drainage of thoracic effusions, nutri-
tional support, anti-inflammatory drugs, and intravenous fluid
therapy. Concurrent treatment with glucocorticoids and anti-
seizure medications may be necessary to reduce the severity of
neurologic signs in dogs with CNS coccidioidomycosis. Topical
prednisolone acetate solution and antiglaucoma agents may be
required for dogs with ocular lesions.
Prognosis
The prognosis for resolution for coccidioidomycosis depends
on the severity of infection and the extent of dissemination. The
duration of treatment ranges from 6 months to many years,
and in some cases, lifelong treatment is needed. IgG antibody
titers decrease with successful treatment, which in general
should be continued until lesions resolve and the titer is 1:2 or
lower. Residual radiographic opacities may be present in some
dogs as a result of scar tissue formation. Dogs with localized
pulmonary infection have the best prognosis; the prognosis
appears to be poor for dogs with CNS involvement. Dogs with
Coccidioides osteomyelitis may require lifelong treatment with
itraconazole.
Immunity and Vaccination
Immunity to Coccidioides spp. depends primarily on cell-
mediated immunity, specifically T lymphocytes. No vaccine is
currently available commercially for prevention of the disease.
Prevention
Prevention of coccidioidomycosis requires avoidance of hyper-
endemic areas (especially parts of Arizona and south-central
California), which may not be possible. In endemic areas, dogs
should be kept away from sites of soil disturbance (such as con-
struction sites) and housed indoors during dust storms; digging
should be discouraged. Housing dogs indoors during the day in
endemic regions may reduce the chance of infection. Neverthe-
less, it should be remembered that clinical infections develop in
only a minority of exposed dogs.
CASE EXAMPLE
Signalment: “Jenny”, a 2-year-old female spayed Labrador
retriever from Oxnard in southwestern California
History: Jenny was evaluated for a 2-month history of
cough, inappetence, and lethargy. For the first 3 days
of illness, the owner noted only an intermittent harsh,
nonproductive cough. On day 4, the dog became lethargic
and inappetent, so she was taken to a local veterinary clinic.
Thoracic radiographs revealed alveolar infiltrates in the left
cranial lung lobe. A diagnosis of bacterial pneumonia was
suspected, so Jenny was treated with enrofloxacin (6 mg/
kg PO q24h), amoxicillin-clavulanic acid (12.5 mg/kg PO
q12h), and metronidazole (8 mg/kg PO q12h) for 2 weeks.
After the first week of treatment, there was no improvement,
CHAPTER 63  Coccidioidomycosis 621
Public Health Aspects
Coccidioidomycosis is a serious infection in human patients and
has similar clinical manifestations to those described in dogs.
The incidence of human coccidioidomycosis has increased in
Arizona in the last decade, possibly as a result of population
growth, immigration of immunologically naïve people, and con-
struction. Construction and agricultural workers, archeologists,
and excavators are at high risk for coccidioidomycosis.2 African
American and Filipino patients, as well as patients with AIDS
and women in the third trimester of pregnancy, are at highest
risk for disseminated disease, which occurs in approximately
0.5% of infected humans.
Because of their susceptibility to infection, dogs have been
used as a sentinel for human exposure to environmental sources
of Coccidioides spp. in seroprevalence studies.34 Direct trans-
mission between infected veterinary patients and humans has
not been reported. However, fatal coccidioidomycosis occurred
in a veterinary resident after necropsy of a horse with dissemi-
nated disease.35 Inoculation coccidioidomycosis was reported
in a veterinary technician from Arizona after a bite from a cat
that was later diagnosed with disseminated disease.23 Inocula-
tion coccidioidomycosis also has the potential to occur if sharps
injuries occur during aspiration of affected tissues, surgical pro-
cedures, or necropsy examinations. Proper sedation or anesthe-
sia should always be used if coccidioidomycosis is suspected in
dogs or cats seen at veterinary clinics and the potential for a
sharps injury exists. If accidental inoculation occurs, immediate
attention should be sought from a physician. Prolonged fluco-
nazole prophylaxis is generally recommended with monitoring
of Coccidioides serology and liver function tests. Bandaging
of skin lesions should be avoided. Necropsies should be per-
formed without delay with suitable protective clothing, and the
carcasses of animals that die from coccidioidomycosis should
immediately be incinerated, because conversion of the organism
to a mycelial form can occur after death of an animal. For the
same reason, burial is not recommended because of the poten-
tial for contamination of the environment. Fluconazole prophy-
laxis is also indicated after accidental laboratory exposure to
inocula.36
so a long-acting cephalosporin was administered (cefovecin,
8 mg/kg SC once). Two days later, treatment with prednisone
(0.3 mg/kg PO q12h) was initiated, which was followed
by mild improvement in the clinical signs. Jenny’s owner
then sought a second opinion on day 20 after onset of
clinical signs, and Coccidioides serology was performed.
The titer was positive for IgG at 1:64. The prednisone
was discontinued, and treatment was initiated with
fluconazole (6.7 mg/kg PO q12h). However, after
1 month of treatment, thoracic radiographic abnormalities
were unchanged. The fluconazole was substituted with
a compounded itraconazole formulation (5 mg/kg PO
q12h). However, after an additional 2 weeks of treatment,
there had been no further improvement in the cough,
so the owner was referred to the University of California,
Davis.
Continued
622 SECTION 3  Fungal and Algal Diseases
According to the owner, Jenny had a poor appetite for dog food
but was eating home-cooked chicken, rice, and vegetables.
Her activity level had been low. The cough occurred many
times throughout the day and was unrelated to exercise and
eating. Occasionally she had paroxysms of cough with a ter-
minal retch. Other medical history included canine parvovi-
rus infection when she was 8 weeks old and “kennel cough”
when she was 9 months old, which resolved after 10 days.
The owners lived in a semirural area just north of Los Angeles.
The only travel had been to Atascadero, further north on the
California coast. She was the only animal in the household.
There had been ongoing construction around the house
to replace a septic system with a sewer, which began 4 to 6
weeks before the onset of clinical signs.
Physical Examination:
General: Quiet but alert and responsive, hydrated. T = 102.6°F
(39.2°C), HR = 100 beats/min, RR = 24 breaths/min, CRT = 1 s.
Eyes, Ears, Nose, and Throat: No abnormalities were noted.
A dilated fundoscopic examination was unremarkable.
Musculoskeletal: Body condition score was 4/9. The dog was
ambulatory with no lameness or muscle atrophy.
Respiratory: Harsh breath sounds were present in all lung
fields. A harsh cough with a terminal retch was readily
elicited on tracheal palpation.
Cardiovascular, Gastrointestinal, Genitourinary, and Lymph
Nodes: No clinically significant abnormalities were detected.
Imaging Findings: Thoracic radiographs were compared to
the two-view thoracic study performed 2 weeks previously,
which was available for review. The initial radiographs showed
a dense alveolar pattern throughout the left cranial lung
lobe and increased opacity in the region of the hilar lymph
nodes with splaying of the mainstem bronchi (see Figure
63-4, C). These abnormalities had improved slightly in the
10-day recheck interim, but alveolar infiltrates were still
present throughout the left cranial lung lobe. There was a
persistent, mild, diffuse pulmonary interstitial pattern in the
remaining pulmonary parenchyma, which was most severe
in the caudodorsal lung fields. This was considered excessive
for the age of the patient. The cardiovascular structures were
within normal limits.
Microbiologic Testing: Coccidioides serology: positive
by qualitative gel immunodiffusion for complement-
fixing antibody (IgG), negative for IgM. Quantitative
immunodiffusion revealed an IgG titer of 1:128.
Serum itraconazole concentration: <0.31 µg/mL (undetectable).
Diagnosis: Pulmonary coccidioidomycosis.
SUGGESTED READINGS
Greene RT, Troy GC. Coccidioidomycosis in 48 cats: a retrospective
study (1984-1993). J Vet Intern Med. 1995;9:86-91.
Johnson LR, Herrgesell EJ, Davidson AP, et al. Clinical, clinicopatho-
logic, and radiographic findings in dogs with coccidioidomycosis: 24
cases (1995-2000). J Am Vet Med Assoc. 2003;222:461-466.
Shubitz LE, Butkiewicz CD, Dial SM, et al. Incidence of Coccidioides
infection among dogs residing in a region in which the organism is
endemic. J Am Vet Med Assoc. 2005;226:1846-1850.
Treatment: The itraconazole capsules prescribed at the
previous veterinary clinic were examined and found to
contain a white powder rather than granules. Treatment was
changed to brand-name itraconazole (Sporanox), because
compounded formulations can be unstable. Instructions
were given to administer the itraconazole (5 mg/kg PO
q24h) with food to improve absorption. Aspirin was also
prescribed (12 mg/kg PO q12h). One month later Jenny’s
cough and activity level had improved considerably. A CBC
was within normal limits, and a biochemistry panel showed
hypoalbuminemia (3.3 g/dL, reference range 3.4-4.3 g/dL)
and hyperglobulinemia (3.4 g/dL, reference range 1.7-
3.1 g/dL). Liver enzyme activities were within reference
ranges. Thoracic radiographs showed resolution of
hilar lymphadenopathy with persistence of the alveolar
infiltrates in the left cranial lung lobe. The IgG antibody
titer to Coccidioides was 1:64. Three months later, the cough
had resolved completely, and after 6 months of treatment,
there was complete resolution of thoracic radiographic
abnormalities and the titer was 1:32. At this point, the owner
switched to a generic formulation of itraconazole because
of financial concerns. After an additional 3 and 6 months of
treatment, the IgG titer was 1:16 and 1:8, respectively, with
no further clinical signs. Treatment was continued for an
additional 3 months (i.e., total of 15 months of treatment),
and the dog remains alive and apparently healthy at the
time of writing.
Comments: This is an interesting case of pulmonary
coccidioidomycosis that occurred outside the hyperendemic
central valley region in California. Exposure may have
resulted from soil disturbance as a result of sewer work.
Because of the low suspicion for the disease, the disease
was mistaken for bacterial pneumonia. The owner had
limited financial resources, so an extensive respiratory work-
up (including respiratory lavage with cytology and culture)
was declined. When Coccidioides serology was positive,
fluconazole was prescribed, but there was no improvement.
The decision to treat with compounded itraconazole
was made because of client financial limitations, but
serum drug levels with this treatment were undetectable.
Fortunately, disease ultimately resolved with a change to a
noncompounded formulation, and there was progressive
decline in serial IgG titers (which were always performed by
the same laboratory). Whether treatment beyond complete
resolution of the radiographic abnormalities was necessary
is unknown.
REFERENCES
1. Hirschmann JV. The early history of coccidioidomycosis: 1892-
1945. Clin Infect Dis. 2007;44:1202-1207.
2. Thompson 3rd GR. Pulmonary coccidioidomycosis. Semin Respir
Crit Care Med. 2011;32:754-763.
3. Ramani R, Chaturvedi V. Antifungal susceptibility profiles of Coc-
cidioides immitis and Coccidioides posadasii from endemic and
non-endemic areas. Mycopathologia. 2007;163:315-319.
4. Fisher MC, Koenig GL, White TJ, et al. Molecular and phenotypic
description of Coccidioides posadasii sp. nov., previously recog-
nized as the non-California population of Coccidioides immitis.
Mycologia. 2002;94:73-84.

5. Flynn NM, Hoeprich PD, Kawachi MM, et  al. An unusual
outbreak of windborne coccidioidomycosis. N Engl J Med.
1979;301:358-361.
6. Schneider E, Hajjeh RA, Spiegel RA, et  al. A coccidioidomycosis
outbreak following the Northridge, Calif, earthquake. JAMA.
1997;277:904-908.
7. Park BJ, Sigel K, Vaz V, et al. An epidemic of coccidioidomycosis in
Arizona associated with climatic changes, 1998-2001. J Infect Dis.
2005;191:1981-1987.
8. Maddy KT. Disseminated coccidioidomycosis of the dog. J Am Vet
Med Assoc. 1958;132:483-489.
9. Johnson LR, Herrgesell EJ, Davidson AP, et al. Clinical, clinicopath-
ologic, and radiographic findings in dogs with coccidioidomycosis:
24 cases (1995-2000). J Am Vet Med Assoc. 2003;222:461-466.
10. Davidson AP, Pappagianis D. Canine coccidioidomycosis: 1970 to
1993. Stanford University, CA: 5th International Conference on
Coccidioidomycosis; 1994:155-162.
11. Butkiewicz CD, Shubitz LE, Dial SM. Risk factors associ-
ated with Coccidioides infection in dogs. J Am Vet Med Assoc.
2005;226:1851-1854.
12. Greene RT, Troy GC. Coccidioidomycosis in 48 cats: a retrospec-
tive study (1984-1993). J Vet Intern Med. 1995;9:86-91.
13. Beaudin S, Rich LJ, Meinkoth JH, et al. Draining skin lesion from
a desert poodle. Vet Clin Pathol. 2005;34:65-68.
14. Shubitz LE, Butkiewicz CD, Dial SM, et al. Incidence of Coccidioi-
des infection among dogs residing in a region in which the organ-
ism is endemic. J Am Vet Med Assoc. 2005;226:1846-1850.
15. Millman TM, O’Brien TR, Suter PF, et  al. Coccidioidomyco-
sis in the dog: its radiographic diagnosis. J Am Vet Radiol Soc.
1979;20:50-65.
16. Burtch M. Granulomatous meningitis caused by Coccidioides
immitis in a dog. J Am Vet Med Assoc. 1998;212:827-829.
17. Pryor Jr WH, Huizenga CG, Splitter GA, et  al. Coccidioi-
des immitis encephalitis in two dogs. J Am Vet Med Assoc.
1972;161:1108-1112.
18. Angell JA, Merideth RE, Shively JN, et al. Ocular lesions associated
with coccidioidomycosis in dogs: 35 cases (1980-1985). J Am Vet
Med Assoc. 1987;190:1319-1322.
19. Shively JN, Whiteman CE. Ocular lesions in disseminated coccidi-
oidomycosis in 2 dogs. Pathol Vet. 1970;7:1-6.
20. Heinritz CK, Gilson SD, Soderstrom MJ, et al. Subtotal pericardectomy
and epicardial excision for treatment of coccidioidomycosis-induced
effusive-constrictive pericarditis in dogs: 17 cases (1999-2003). J Am
Vet Med Assoc. 2005;227:435-440.
21. Angell JA, Shively JN, Merideth RE, et al. Ocular coccidioidomy-
cosis in a cat. J Am Vet Med Assoc. 1985;187:167-169.
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22. Tofflemire K, Betbeze C. Three cases of feline ocular coccidioido-
mycosis: presentation, clinical features, diagnosis, and treatment.
Vet Ophthalmol. 2010;13:166-172.
23. Gaidici A, Saubolle MA. Transmission of coccidioidomycosis to a
human via a cat bite. J Clin Microbiol. 2009;47:505-506.
24. Sykes JE. Unpublished observations, 2012.
25. Gershman N, Villaba W, Neel K. Detection of canine IgG and IgM
antibodies to Coccidioides immitis using a novel enzyme-linked
immunosorbent assay (EIA) method. Stanford University, CA: 5th
International Conference on Coccidioidomycosis; 1994:137-146.
26. Graupmann-Kuzma A, Valentine BA, Shubitz LF, et  al. Coccidi-
oidomycosis in dogs and cats: a review. J Am Anim Hosp Assoc.
2008;44:226-235.
27. Greene RT. Coccidioidomycosis and paracoccidioidomycosis. In:
Greene CE, ed. Infectious Diseases of the Dog and Cat. 4th ed.
St. Louis, MO: Elsevier Saunders; 2012:634-645.
28. Yturraspe DJ. Clinical evaluation of a latex particle agglutination
test and a gel diffusion precipitin test in the diagnosis of canine coc-
cidioidomycosis. J Am Vet Med Assoc. 1971;158:1249-1256.
29. Kirsch EJ, Greene RT, Prahl A, et  al. Evaluation of Coccidioides
antigen detection in dogs with coccidioidomycosis. Clin Vaccine
Immunol. 2012;19:343-345.
30. Vucicevic D, Blair JE, Binnicker MJ, et al. The utility of Coccidioi-
des polymerase chain reaction testing in the clinical setting. Myco-
pathologia. 2010;170:345-351.
31. Lauer A, Baal JD, Baal JC, et al. Detection of Coccidioides immi-
tis in Kern County, California, by multiplex PCR. Mycologia.
2012;104:62-69.
32. Reed RE, Hoge RS, Trautman RJ. Coccidioidomycosis in two cats.
J Am Vet Med Assoc. 1963;143:953-956.
33. Galgiani JN, Catanzaro A, Cloud GA, et  al. Comparison of oral
fluconazole and itraconazole for progressive, nonmeningeal coc-
cidioidomycosis. A randomized, double-blind trial. Mycoses Study
Group. Ann Intern Med. 2000;133:676-686.
34. Gautam R, Srinath I, Clavijo A, et al. Identifying areas of high risk
of human exposure to coccidioidomycosis in Texas using serology
data from dogs. Zoonoses Public Health, 2012.
35. Kohn GJ, Linne SR, Smith CM, et  al. Acquisition of coccidioi-
domycosis at necropsy by inhalation of coccidioidal endospores.
Diagn Microbiol Infect Dis. 1992;15:527-530.
36. Stevens DA, Clemons KV, Levine HB, et al. Expert opinion: what
to do when there is Coccidioides exposure in a laboratory. Clin
Infect Dis. 2009;49:919-923.
CHAPTER 64
Sporotrichosis
Jane E. Sykes
Overview of Sporotrichosis
First Described: 1896, in Maryland, USA, by Benjamin Schenk1
Cause: Sporothrix schenckii complex fungi (ascomycetes, tele-
omorph Ophiostoma stenoceras)
Affected Hosts: Cats, dogs, humans, and a variety of other
mammalian and avian species
Geographic Distribution: Tropical and temperate zones
worldwide
Mode of Transmission: Cutaneous inoculation or inhalation
of conidia
Major Clinical Signs: Cutaneous nodules or draining skin
lesions, lymphadenopathy
Differential Diagnoses: Other deep mycoses (especially
cryptococcosis in cats), mycobacterial infections, nocar-
diosis or actinomycosis, leishmaniosis, squamous cell
carcinoma, feline eosinophilic granuloma complex, feline
herpesviral dermatitis, foreign body granulomas
Human Health Significance: Many human infections are
acquired from the environment, but direct transmission
can follow contact between infected cats and humans,
and especially cat bites or scratches.
Etiology and Epidemiology
Organisms that belong to the Sporothrix schenckii species com-
plex are dimorphic, saprophytic fungi that cause sporotrichosis,
a subacute to chronic disease that most often results from cuta-
neous or subcutaneous inoculation of the organism through a
puncture wound, but can also follow inhalation of the fungus.
Sporotrichosis has been described in cats, dogs, humans, and
a variety of other domestic and wild animal hosts that include
equine species, ruminants, swine, rats, mice, hamsters, armadil-
los, domestic fowl, chimpanzees, and dolphins.
Sporothrix species thrive in humid conditions (92% to 100%)
and at mean temperatures between 25°C and 30°C. They prefer
moist soil that is rich in decaying vegetation, as well as sphag-
num moss, wood, thorns, or hay, where they grow as mycelia.
The fungus is distributed worldwide in tropical and temperate
zones but is most prevalent in tropical or subtropical regions
of the Americas. Regions of endemicity include Mexico, Brazil,
Uruguay, Peru, Japan, India, and South Africa; sporotrichosis
is uncommon in Europe. Although all Sporothrix isolates have
in the past been grouped into one species, multiple genotypes
exist that vary in virulence and geographic distribution.2-4 As a
624
result, an S. schenckii species complex has been identified, which
includes at least six species: S. schenckii sensu stricto, Sporothrix
brasiliensis, Sporothrix globosa, Sporothrix mexicana, Sporo-
thrix albicans, and Sporothrix luriei.5-7 The epidemiology of the
various members of the complex is as yet incompletely under-
stood. S. globosa has a worldwide distribution, whereas S. brasil-
iensis appears to be limited to Brazil. S. schenckii, S. brasiliensis,
and S. albicans have been isolated from cats, and S. schenckii and
S. luriei have been isolated from dogs.8
Cats are more susceptible to sporotrichosis than dogs and
are thought to facilitate spread of organisms in the environ-
ment.9 Contaminated claw or bite wounds, as well as autoinoc-
ulation during grooming, are thought to be important modes of
transmission.10 Male cats are overrepresented in most studies
(60% to 70% of affected cats), which may reflect the tendency
of male cats to roam outdoors and be involved in aggressive
interactions.10-13 In Rio de Janeiro, a large outbreak of sporo-
trichosis has occurred over the past decade, which has involved
more than 1000 humans, 60 dogs, and 1500 cats.1 Of affected
cats in this outbreak, Sporothrix spp. were isolated from 100%
of 76 cutaneous lesions, 66% of 71 swab specimens from the
nasal cavity, 41% of 79 oral swab specimens, and 40% of 38
nail fragments.14 The fungus was also isolated from 3 of 84
apparently healthy cats that were in contact with affected ani-
mals. Twenty percent of cats were positive for antibodies to
FIV, 1% of cats were positive for FeLV antigen, and 1% of
cats were positive for both FIV antibodies and FeLV antigen.10
More than 85% of affected cats were less than 4 years of age.
Of isolates from affected humans in the outbreak, most were
S. brasiliensis, but S. schenckii sensu stricto and S. globosa were
also isolated.7
In dogs, sporotrichosis is relatively rare. Cutaneous intro-
duction of the organism with plant foreign bodies has been sug-
gested in some cases. In one dog, the portal of entry was thought
to be an alligator bite wound.15 In the Brazilian outbreak, con-
tact with cats appeared to be an important mode of transmission
to dogs.16 No clear breed or sex predisposition has been identi-
fied, and dogs in the Brazilian outbreak ranged in age from 6
months to 12 years (median, 4 years). Mixed sporotrichosis and
cryptococcosis was reported in a dog from Ontario.17
Clinical Features
Signs and Their Pathogenesis
Transmission of Sporothrix primarily results from cutane-
ous inoculation of organisms in the environment, but can also
result from inhalation of conidia. Direct contact with cats that
have sporotrichosis (without an apparent break in the skin) can
also result in transmission.10 Once within tissues, the organism
 CHAPTER 64  Sporotrichosis 625

TABLE 64-1
Proposed Classification Scheme for Clinical Forms
of Sporotrichosis in Human Patients1
Relevance to Cats
Group Subcategory and Dogs
Cutaneous Fixed (or localized) All forms described in
Lymphocutaneous cats and dogs, but
Multifocal (or dis- fixed or ­multifocal
seminated) disease most ­common
Mucosal Ocular Occurs in cats and
Nasal dogs
Other
Extracutaneous Pulmonary Pulmonary and gen-
Osteoarticular eralized disease is
Meningeal primarily described
Generalized (or in cats. Pulmonary,
disseminated) generalized, and os-
teoarticular disease
occur rarely in dogs.
A
Special forms Spontaneous Spontaneous regression
­regression has been described
Hypersensitivity in cats and especially
(erythema nodo- dogs
sum, erythema
multiforme)

converts into a yeast form. The yeast form has a cell wall that
contains glucans, a glycoprotein fraction known as peptidor-
hamnomannan, and galactose-containing polysaccharides. The
cell wall also contains melanin, which may protect the yeast
from oxidative damage.1,9 Adhesins on the surface of the organ-
ism bind fibronectin, which appears to be important for viru-
lence.18 Organisms that are thermotolerant (able to survive at
temperatures closer to body temperature, or 37°C) may be more
likely to cause invasive disease.9
Several clinical forms of sporotrichosis are recognized in
human patients. The clinical form of disease that develops
depends on host factors and the virulence of the infecting Spo-
rothrix strain. A suggested classification system for human
sporotrichosis categorizes the distribution of lesions into cuta-
neous, mucosal, or extracutaneous (Table 64-1). Cutaneous
lesions are crusted, plaque-like, or nodular lesions that often
ulcerate or drain serosanguineous fluid. They may be fixed
(localized), track along the course of lymphatics (lymphocuta-
neous), or be multifocal (sometimes referred to as disseminated
cutaneous disease) (Figures 64-1 and 64-2). More than 50%
of human patients develop the lymphocutaneous form and the
infection spreads proximally along lymphatics, with formation
of multiple secondary nodules (also known as gummata) that
may ulcerate. This form has not been clearly recognized in cats
and appears to be uncommon to rare in dogs.16 Up to 80%
of cats with sporotrichosis have multiple, widely distributed
cutaneous lesions.10,13 These may develop as a result of auto-
inoculation during grooming, or they can be a manifestation
of a disseminated (or generalized) systemic infection.
Extracutaneous disease follows inhalation of conidia or
hematogenous spread of Sporothrix from sites of cutaneous
B
FIGURE 64-1  Cutaneous and mucosal sporotrichosis. A, 2-year-old female spayed
Bengal that had ulcerated lesions of the face, thoracic limb, and tail. B, Cutaneous and
conjunctival sporotrichosis in a 1-year-old male neutered domestic shorthair. (Courtesy
University of California, Davis, Veterinary Dermatology Service.)
inoculation. Transmission by inhalation is thought to occur in
some cats, because pulmonary and nasal cavity disease with
associated respiratory signs are common in cats.10-13,19 Respira-
tory signs include sneezing, cough, tachypnea, increased respi-
ratory effort, stertor, and/or nasal discharge. Respiratory signs
were present in 44% of 347 cats in the Brazilian outbreak10 and
can also occur in dogs.13,16 Sneezing may occur before the onset
of cutaneous lesions in cats, and some cats or dogs show only
respiratory signs. Cats also can develop generalized disease, with
involvement of multiple organ systems, which include the lungs,
liver, spleen, kidneys, lymph nodes, and testicles.13,20 Co-infection
with FIV does not appear to be a risk factor for dissemination.21
Isolation of Sporothrix species from the blood of 35% of 49 cats
with focal or multifocal cutaneous involvement was reported.21
Clinical signs of systemic involvement include fever, generalized
626 SECTION 3  Fungal and Algal Diseases
A B
FIGURE 64-2  Multifocal ulcerated cutaneous lesions on the head (A) and left thoracic limb (B) caused by Sporothrix spp. in a 2-year-old male neutered Labrador retriever. (Courtesy
University of California, Davis, Veterinary Dermatology Service.)
TABLE 64-2
Diagnostic Assays Currently Available for Sporotrichosis in Dogs and Cats
Assay Specimen Type Target
Cytologic Aspirates of cutaneous nodules, Sporothrix yeasts High sensitivity (approximately 80%) in cats. Sensitivity is
examination impression smears of skin
lesions or biopsies
Fungal culture Aspirates, swabs, or biopsies of Sporothrix spp.
affected tissues
Histopathology Biopsy specimens from affected Sporothrix spp.
tissues yeasts
lymphadenomegaly, anorexia, dehydration, vomiting, and
weight loss. These signs were each reported in fewer than 20%
of cats with sporotrichosis; vomiting occurred in fewer than 5%
of the cats.10 Weight loss, vomiting, or anorexia can also occur
in dogs.13,16 Other rare forms of extracutaneous sporotrichosis
described in humans include osteoarticular and meningeal sporo-
trichosis. Osteoarticular sporotrichosis occurs rarely in dogs and
may manifest clinically as lameness and synovial effusion.12,22
Physical Examination Findings
In cats, cutaneous lesions are most often found on the head
(especially the bridge of the nose and the nasal planum, eyelids,
and/or pinnae) and have a similar appearance to those caused
by Cryptococcus spp. (see Figure 64-1).10,12,23 Other common
sites for skin lesions are the distal limbs, digits, and tail, but
lesions can also be found elsewhere on the body. Swelling of
the conjunctiva or lesions in the oral cavity or nasal cavity can
be present in some affected cats (see Figure 64-1). Cutaneous
or subcutaneous lesions are often firm nodules that sometimes
enlarge to form soft cutaneous masses. Nodules can ulcerate
and drain serosanguineous to purulent fluid, but are usually not
painful or pruritic.12 In some cases, ulcerations and draining
skin lesions are not associated with the formation of discrete
Performance
lower in dogs.
Generally sensitive (around 75%), but false negatives can
occur if specimen size is low. Most specific method.
Requires several weeks’ incubation. Allows species
identification via molecular methods and antifungal
drug susceptibility testing.
Sensitivity is higher (>60%) in cats when compared with
dogs (<20%). Special stains such as Gomori’s methena-
mine silver and immunohistochemical stains facilitate
organism detection and identification.
nodules. Lesions range in diameter from a few millimeters to
several centimeters. Extensive zones of necrosis with exposure
of underlying muscle and bone have also been described.10
Regional lymphadenomegaly may be detected. Other less fre-
quent physical examination findings in cats are fever, nasal
discharge, decreased nasal airflow, tachypnea, increased respi-
ratory effort, dehydration, and thin body condition.
In dogs, lesions consist of single or multiple ulcers or nodules
on the head, trunk, forelimbs, and/or digits (see Figure 64-2).
Rarely, they course along lymphatics.12,13,16,24 The nasal pla-
num was affected in 59% of 44 dogs in one study, and 21% of
dogs had nasal mucosal involvement.16 Regional lymphadeno-
megaly is common.
Diagnosis
Diagnosis of sporotrichosis rests on identification of the organ-
ism by cytologic or histopathologic evaluation, or by fungal
culture of lesions (Table 64-2). A high degree of suspicion is
required for the disease in non-endemic regions. Sporotrichosis
should be considered in any dog or cat with cutaneous lesions
suggestive of cryptococcosis or other deep mycoses, and espe-
cially any cat with ulcerative or nodular lesions of the face.

B
FIGURE 64-3  Nasal sporotrichosis with communication to the overlying skin in a
14-year-old male neutered domestic shorthair. A, The cat had a 1-year history of sneezing and
a 2-month history of a mass on the bridge of the nose. B, Computed tomography revealed a
nasal cavity mass with nasal bone destruction and involvement of the subcutaneous tissues.
Laboratory Abnormalities
Complete Blood Count
In many dogs and cats with sporotrichosis, the CBC is unre-
markable. The most common abnormalities are mild anemia
and leukocytosis due to a neutrophilia, sometimes with a mild
left shift.10,24 Monocytosis, eosinophilia, lymphocytosis, or
lymphopenia may also be present. Approximately 50% of dogs
in the Brazilian outbreak were anemic, and around 20% had
eosinophilia or neutrophilia.16
Serum Biochemical Tests
The most frequent serum biochemistry findings in cats and dogs
with sporotrichosis are hyperglobulinemia and hypoalbumin-
emia.10,16 Rarely, increased liver enzyme activities are present
in affected dogs.16
CHAPTER 64  Sporotrichosis 627
FIGURE 64-4  Cytology of an impression smear from a skin lesion of the cat in Figure
64-1, A. There are large numbers of extracellular and intracellular pleomorphic yeasts, with
morphology consistent with Sporothrix spp.
Urinalysis
Urinalysis in animals with sporotrichosis usually reveals no clin-
ically significant abnormalities.
Diagnostic Imaging
Plain Radiography
Findings on plain thoracic radiographs in cats with pulmonary
sporotrichosis have not been described in detail. An interstitial
infiltrate was described in one affected cat.20 Thoracic radio-
graphs were performed on one dog and five cats seen at the
author’s teaching hospital, but no radiographic abnormalities
were present. Radiographs of bones that underlie skin lesions
typically reveal only soft tissue swelling; rarely is underlying
osteomyelitis detected.
Advanced Imaging
In the author’s hospital, computed tomography (CT) of the
nasal cavity in a cat with a lesion on the bridge of the nose
revealed an irregular rim-enhancing mass within the nasal cavity
that invaded through the nasal bones to involve the subcutane-
ous tissues (Figure 64-3). Severely thickened turbinate mucosa,
an intranasal soft tissue density mass, and focal osteolysis were
reported on nasal CT in a dog with signs of nasal cavity disease
due to sporotrichosis.25
Microbiologic Tests
Cytologic Examination
Cytologic examination of impression smears or aspirates of skin
lesions typically reveals pyogranulomatous inflammation. In
cats, large numbers of round to cigar-shaped yeasts can be found
intracellularly within neutrophils or macrophages and extracel-
lularly (Figure 64-4). The yeasts are 4 to 6 µm in diameter and
can exhibit a single bud with a narrow base. The sensitivity of
cytologic examination of impression smears from skin lesions of
806 cats with sporotrichosis from Brazil was 79%.26 Yeasts are
usually not found in bronchoalveolar lavage fluid from cats with
respiratory signs.19 In general, dogs have very low numbers of
628 SECTION 3  Fungal and Algal Diseases

A B C
FIGURE 64-5  Sporothrix schenckii growing on laboratory media. A, The fungus grows on fungal isolation media as a white mold at 30°C with production of pigmented conidia.
B, Using light microscopy, the mold form is characterized by the production of conidia in a flower-like arrangement. Lactophenol cotton blue stain, 1000× oil magnification. C, At 37°C,
the organism converts to the yeast form. Gram stain, 1000× oil magnification.

organisms in skin lesions, which leads to a high proportion of

false-negative results.12,16 Sporothrix spp. may be confused with
Histoplasma capsulatum, Cryptococcus spp., Candida spp.,
Leishmania infantum, and potentially other protozoan parasites
such as Toxoplasma gondii and Neospora caninum.

Fungal Culture
Definitive diagnosis of sporotrichosis can be made by isola-
tion and identification of the organism from clinical speci-
mens (such as skin biopsies, blood, nasal swabs, or respiratory
lavage specimens), preferably in both mycelial and yeast forms.
The sensitivity of isolation from skin biopsies exceeds 75% in
both dogs and cats.10,13,16 The chance of successful culture is
reduced if swab specimens of exudate instead of biopsies are
submitted. The organism grows in culture as a white mold,
which becomes brown to black as a result of production of
pigmented conidia, which are arranged in florets on a stem
(conidiophore) (Figure 64-5). Although growth can occur in
5 to 7 days, sometimes several weeks of incubation may be
required.9 When the organism is incubated on rich media such
as brain-heart infusion at 37°C, it converts to oval to cigar-
shaped yeasts. The organism can then be identified based on
light microscopic appearance of the mold and yeast forms (see
Figure 64-5, B and C). Determination of the Sporothrix species
present requires subsequent molecular analysis by specialized
laboratories.27 Antifungal susceptibility testing can be per-
formed, but established breakpoints are not available. Never-
theless, it may be of help for treatment of refractory cases (see
Case Example).
Serologic Diagnosis
ELISA assays that detect antibody to Sporothrix cell wall anti-
gens have been developed for diagnosis of human and feline
sporotrichosis.28,29 These assays are not widely available for
diagnosis of sporotrichosis in cats or dogs. Currently, no anti-
gen assays are available, but cross-reactivity with the H. capsu-
latum antigen assay occurs in human patients.30
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays have been described for detection of Sporothrix spp.
in lesions.31,32 These have not been widely applied to the diag-
nosis of sporotrichosis in cats or dogs.
FIGURE 64-6  Histopathology of a biopsy from the skin of an 11-year-old female
spayed domestic shorthair cat. Granulomatous inflammation is present with abundant
intracytoplasmic fungal organisms consistent with Sporothrix spp.
Pathologic Findings
Gross pathologic findings at necropsy in cats with generalized
sporotrichosis include multifocal, pinpoint (1 mm) white nodu-
lar lesions within the lungs and lymphadenomegaly.20 Histopa-
thology of lesions from cats usually reveals pyogranulomatous
inflammation with a variable number of neutrophils, histio-
cytes, multinucleated giant cells, and lymphocytes, with fewer
plasma cells, eosinophils, and mast cells (Figure 64-6).12,13
Organized granuloma formation, with focal accumulations of
macrophages and sometimes an outer layer of plasma cells, is
found in a minority (<15%) of skin biopsies from affected cats10
and in more than 30% of skin biopsies from affected dogs.16,33
Intracellular and extracellular yeasts are seen on histopathology
in more than 60% of skin biopsies from affected cats.10,13 In
contrast, organisms were found in biopsy specimens from fewer
than 20% of dogs with sporotrichosis.16,33 The presence of the
Splendore-Hoeppli phenomenon (“asteroid bodies”) around
yeasts is commonly described in human infections, but this is
not typically observed in dogs or cats.9 Application of stains
such as periodic acid–Schiff (PAS) and especially Gomori’s

methenamine silver (GMS) increase the chance that organisms
are detected.12,34 However, despite the use of such stains, diag-
nosis of canine sporotrichosis can be frustrating because of the
scarcity of organisms in tissues. The use of immunohistochemis-
try (IHC) to aid histologic diagnosis has been reported, although
this is not readily available.34 In one study, the sensitivity of
PAS, GMS, and IHC for detection of Sporothrix spp. in dogs
with sporotrichosis was 20%, 44%, and 66%, respectively.34
The use of the stains in combination increased the sensitivity to
over 80%.
Treatment and Prognosis
Antimicrobial Treatment
The treatment of choice for sporotrichosis in dogs, cats, and
humans is itraconazole (see Chapter 9 for recommended doses).
In human patients, use of itraconazole results in cure rates for
cutaneous sporotrichosis that exceed 90%.35 Itraconazole resis-
tance has been documented in some Sporothrix isolates from
animals.8 Fluconazole is considered less active than itraconazole
for treatment of human sporotrichosis but was used successfully
to treat disseminated disease in a cat.12 Treatment of human,
canine, and feline sporotrichosis with ketoconazole is more
likely to result in relapses, and development of drug toxicity
may limit therapy.10,12,22 In affected cats from Brazil, clinical
cure was 1.3 times more likely in cats treated with itraconazole
than in cats treated with ketoconazole.11
Alternative antifungal drugs for animals with refractory dis-
ease include supersaturated potassium or sodium iodide, terbin-
afine, or amphotericin B. Oral administration of supersaturated
potassium iodide (SSKI) for 30 days beyond complete resolu-
tion of clinical signs was used to treat sporotrichosis in cats,
dogs, or humans before the widespread availability of itracon-
azole. However, treatment failures and toxic side effects that
include anorexia, vomiting, diarrhea, and lethargy are common
(41% of cats in one recent report).10,22,36,37 It has been recom-
mended that the use of SSKI be reserved for fixed cutaneous,
lymphocutaneous, or mucosal forms of the disease in human
patients. Because of its extremely bitter taste, potassium iodide
(KI) capsules have also been used to treat affected cats, with a
cure rate of 48% after 4 to 5 months.23 Inappetence occurred in
52% of cats, and a smaller percentage of cats developed weight
loss, diarrhea, or vomiting. Increased liver enzyme activities can
also occur. The dose range used was 2.5 to 20 mg/kg PO q24h
(median, 15 mg/kg). The dose was increased in 2.5 mg/kg incre-
ments every 5 days from an initial dose of 5 mg/kg PO q24h.
Alterations in thyroid function can be an adverse effect of KI
treatment in humans.35 Treatment with terbinafine was success-
ful in a human with nasal sporotrichosis that failed treatment
with KI and itraconazole.38
Although amphotericin B is effective, nephrotoxicity and
the need for parenteral administration have limited its use.
The primary indication for amphotericin B is for treatment of
sporotrichosis that is refractory to itraconazole. Intralesional
amphotericin B administration in combination with itracon-
azole was used successfully to treat fixed or multifocal cutane-
ous lesions in cats that were refractory to itraconazole alone.39
The solution was prepared by addition of 5 mL of 2% lidocaine
and 5 mL of distilled water to a 50 mg vial of amphotericin B
(final concentration, 5 mg/mL). A total of 0.5 to 1.5 mL (median
0.7 mL) was administered intralesionally once a week to once
every other week, and 73% of the cats were cured. The number
CHAPTER 64  Sporotrichosis 629
of treatments administered ranged from 1 to 5 (median, 2 treat-
ments per cat). Sterile abscess formation at the site of adminis-
tration occurred in 15% of cats.
Lastly, localized hyperthermia has been used as a safe and
inexpensive method to treat fixed cutaneous and lymphocutane-
ous sporotrichosis in human patients and was used as the sole
treatment in a cat with localized cutaneous disease.40 In that
case, a thermal bag that reached a temperature of 40°C to 42°C
was applied twice daily for 15 minutes. When used alone, this
method is not likely to be effective in animals with disseminated
disease. Surgery or cryotherapy are other alternatives suggested
for treatment of localized sporotrichosis in human patients.35
Prognosis
Treatment durations of 4 to 6 months are usually required
for successful treatment of sporotrichosis. A median time to
clinical cure of 26 weeks (range, 8 to 131 weeks) was reported
for 175 cats treated with itraconazole.11 Treatment should be
continued for a month after lesions resolve. Some cats may
require more than a year of antifungal drug treatment.10 In
general, the prognosis for cure of sporotrichosis is good, with
more than 70% of cats in some studies cured of the disease,
regardless of the number of skin lesions present.10,12 Lower
cure rates of 38% for itraconazole and 29% for ketoconazole
were reported in another study.11 Co-infection with FIV does
not seem to alter the likelihood of treatment success,10 but the
presence of respiratory signs was associated with a twofold
increase in death rate.11 Relapse (or re-infection) may occur
between 3 and 18 months after discontinuation of antifungal
drug therapy. Spontaneous regression of a persistent cutane-
ous lesion was also described in a cat.10 Euthanasia may result
from expense related to antifungal drug therapy or concerns
for zoonotic transmission.
Spontaneous regression of cutaneous lesions has been
described in 15% of 33 dogs.16 Nearly 80% of dogs were
cured after itraconazole or ketoconazole treatment for 2 to 15
months (median, 2.5 months for itraconazole and 3.5 months
for ketoconazole).
Immunity and Vaccination
Immunity imparted by T cells appears to be important in limit-
ing the extent of sporotrichosis.9,41 There is no vaccine.
Prevention
Indoor housing and neutering may reduce sporotrichosis in cats.
Isolation of affected cats from other cats and dogs while they are
properly treated may also reduce transmission.
Public Health Aspects
Many human sporotrichosis cases result from cutaneous
inoculation of organisms in soil or on plant material. Activi-
ties associated with human sporotrichosis include rose garden-
ing, topiary production, armadillo hunting, mining, carpentry,
forestry work, Christmas tree farming, and hay baling, and
outbreaks have been reported after common exposure of mul-
tiple individuals to a source of contaminated plant material.
Laboratory-acquired infections have also been described.42,43
Zoonotic transmission has followed bites or scratches from
squirrels, horses, dogs, pigs, mules, insects, and especially cats.
630 SECTION 3  Fungal and Algal Diseases
In one study from Malaysia, slightly more human sporotrichosis
cases resulted from cat bites or scratches than from exposure
to plant foreign bodies.44 Direct transmission can occur from
infected cats to humans without an apparent break in the skin.
In the epidemic in Rio de Janeiro, disease in cats preceded that
in humans and dogs. Nearly 85% of affected humans reported
contact with sick cats, and more than half of these individuals
reported a history of a scratch or a bite. Women were more
likely than men to be affected, especially those involved in
domestic activities and animal care.45 Five percent of the cases
were veterinarians or veterinary technicians. Of cats with spo-
rotrichosis from 225 residences in this outbreak, 91 people
who had contact with infected cats acquired the infection.10
In contrast, no human cases were associated with a series of
CASE EXAMPLE
Signalment: “Bingo,” a 2-year-old male neutered domestic
longhair cat from Fairfield in northern California
History: Bingo was referred to the University of California,
Davis, Veterinary Medical Teaching Hospital for evaluation
of refractory cryptococcosis. Initially Bingo’s owners noted
signs of snuffling, sneezing, and nasal discharge, which they
attributed to a “cold.” One month later a lump appeared on
Bingo’s nose. Bingo was taken to a local veterinary clinic
where cytologic examination of an impression smear of
the mass revealed organisms that were suspected to be
Cryptococcus spp. A CBC showed no abnormalities. The
owners declined treatment with itraconazole because
of financial limitations, so fluconazole was prescribed
(10 mg/kg PO q12h). Despite this treatment, the respiratory
signs persisted, nodular skin lesions appeared on the face,
and mandibular lymphadenopathy developed. Bingo had
been an indoor-outdoor cat but had been housed exclusively
indoors for the previous few months. He lived with five other
indoor cats and a dog, all of which were well. His appetite and
activity level had been normal and he was fed a commercial
dry cat food. According to the owners, Bingo had tested
negative for FeLV and FIV approximately 1 year previously.
A B
FIGURE 64-7  Effect of treatment with itraconazole and amphotericin B in a 2-year-old male neutered domestic longhair cat with multifocal cutaneous and mucosal sporotrichosis.
A, Before institution of treatment. B, After itraconazole and eight treatments with amphotericin B. C, After an additional 7 months of itraconazole treatment.
23 veterinary cases from northern California.12 Generalized
extracutaneous sporotrichosis with involvement of multiple
organs has been described in immunocompromised humans,
such as those with AIDS.46,47
In contrast to feline sporotrichosis, canine sporotrichosis is of
minimal zoonotic importance. As occurs in humans, dogs have
a paucity of organisms within tissues. There were no reports of
transmission to humans as a result of contact with affected dogs
in the Rio de Janeiro outbreak.16
Owners of dogs and cats with sporotrichosis should be told
to minimize contact with affected animals. If contact is nec-
essary, gloves should be worn and proper hand washing per-
formed. Owners should also be informed of the potential for
exposure to organisms in the environment.6
Physical Examination:
Body Weight: 5.7 kg.
General: Bright, alert, responsive, and hydrated. T = 102.2°F
(39.0°C), HR = 140 beats/min, RR = 40 breaths/min, CRT 1 s.
Integument, Ears, Eyes, Nose, and Throat: Ulcerated, firm
cutaneous nodules (1 cm) were present on the left pinna and
first digit of the left thoracic limb (0.25 cm). An ulceration
(0.5 cm) was present on the bridge of the nose, which was
markedly swollen (Figure 64-7, A). There was decreased nasal
airflow bilaterally that was more severe on the left side, and
mild left-sided mucoid nasal discharge. Marked soft tissue
swelling occluded the left nares. There was moderate, left-
sided serous ocular discharge. No lesions were detected on
fundoscopic examination.
Musculoskeletal: Body condition score was 6/9, ambulatory.
Cardiovascular: No clinically significant abnormalities were
detected.
Respiratory: There was a mild increase in respiratory effort
with stertor. Thoracic auscultation revealed referred upper
airway sounds.
Gastrointestinal and Genitourinary: Abdominal palpation
was unremarkable. A small amount of dried feces and a
tapeworm proglottid protruded from the anus.
Lymph Nodes: Only the left mandibular lymph node was
enlarged (2 cm in diameter).
C

Microbiologic Testing: Latex agglutination for Cryptococcus
antigen: Negative.
Cytologic examination of impression smears from the nares:
Erythrocytes, neutrophils, and occasional epithelial cells
were present. No fungal elements were identified.
Histopathology of a biopsy from the nasal cavity: Pyogranulo-
matous inflammation with intralesional fungi with morphol-
ogy consistent with Sporothrix spp.
Diagnosis: Sporotrichosis with nasal and multifocal cutaneous
involvement.
Treatment: Bingo was treated with a compounded form of
itraconazole (5 mg/kg PO q12h). Clinical improvement did
not occur after 2 weeks, so terbinafine was added (30 mg/
kg PO q24h). One month later, two nodules appeared on
the conjunctiva of the left eye, and the lesions on the left
pinna, left thoracic limb, and nasal bridge had enlarged. Mild
subcutaneous emphysema of the face was also apparent
during respiration. The cat was bright and appetent, but
body weight had decreased to 5.1 kg. No abnormalities
were detected on the CBC and serum biochemistry panel.
Large numbers of Sporothrix yeasts were seen on impression
smears of the thoracic limb lesion. Fungal culture yielded
S. schenckii sensu stricto (identified at the University of
Texas Health Science Center at San Antonio based on ITS,
D1/D2, and calmodulin gene sequencing). Antifungal
drug susceptibility testing revealed minimum inhibitory
concentrations of 1, 2, 0.06, greater than 64, 1, 1, and 16 µg/
mL for amphotericin B, 5-flucytosine, terbinafine, fluconazole,
itraconazole, posaconazole, and voriconazole, respectively. A
serum itraconazole concentration was less than 0.31 µg/mL.
Subsequently the cat was hospitalized and treated with
itraconazole (Sporanox) solution (3 mg/kg PO q12h) and
deoxycholate amphotericin B (0.25 mg/kg IV). Terbinafine
treatment was discontinued. Cutaneous lesions were also
treated with local hyperthermia (15 minutes q8h). After
SUGGESTED READINGS
Barros MB, de Almeida Paes R, Schubach AO. Sporothrix schenckii and
sporotrichosis. Clin Microbiol Rev. 2011;24:633-654.
Crothers SL, White SD, Ihrke PJ, et al. Sporotrichosis: a retrospective
evaluation of 23 cases seen in northern California (1987-2007). Vet
Dermatol. 2009;20:249-259.
Vasquez-del-Mercado E, Arenas R, Padilla-Desgarenes C. Sporotricho-
sis. Clin Dermatol. 2012;30:437-443.
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and sporotrichosis. An Acad Bras Cienc. 2006;78:293-308.
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virulence of different Sporothrix schenckii clinical isolates using
experimental murine model. Med Mycol. 2007;45:721-729.
3. Kong X, Xiao T, Lin J, et al. Relationships among genotypes, vir-
ulence and clinical forms of Sporothrix schenckii infection. Clin
Microbiol Infect. 2006;12:1077-1081.
4. Zhang Z, Liu X, Lv X, et  al. Variation in genotype and higher
virulence of a strain of Sporothrix schenckii causing disseminated
cutaneous sporotrichosis. Mycopathologia. 2011;172:439-446.
CHAPTER 64  Sporotrichosis 631
eight amphotericin B treatments, the lesions had halved
in size (Figure 64-7, B). An additional three treatments
were administered, after which a mild increase in BUN
concentration developed (43 mg/dL, reference range 18-33
mg/dL). Serum creatinine was within the reference range
(1.4 mg/dL, reference range 1.1-2.2 mg/dL). The serum
itraconazole concentration was 4.03 µg/mL. A new lesion
appeared on the right distal thoracic limb, and impression
smears of the lesion revealed large numbers of Sporothrix
yeasts. The itraconazole dose was increased to 5 mg/kg PO
q12h, but was reduced by the owner to 5 mg/kg PO q48h after
5 months because Bingo’s appetite was slightly decreased.
When the cat was reevaluated 7 months after completion of
amphotericin treatment, lesions had improved dramatically
(Figure 64-7, C). The owner was encouraged to increase the
dose of itraconazole to 5 mg/kg PO q24h, but the cat was
subsequently lost to follow-up.
Comments: This cat had nasal and multifocal cutaneous
sporotrichosis that was somewhat refractory to treatment
with antifungal drugs. The disease was initially mistaken
for cryptococcosis. Limited client financial resources and
compliance may have influenced treatment outcome in this
case; costs of fungal culture, identification, and antifungal
susceptibility testing were not charged to the client.
Fluconazole was ineffective, and the antifungal susceptibility
panel suggested resistance to this azole. The use of
compounded itraconazole resulted in suboptimal serum drug
concentrations. However, once treatment with amphotericin B,
a brand name itraconazole formulation, and heat compression
of lesions was instituted, gradual clinical improvement
occurred. The change in itraconazole formulation alone might
have been sufficient to cause disease resolution. The owners
were told to wear gloves when handling the cat and to wash
their hands afterward. No disease was reported in the owners
or any other animals in the household.
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6. Marimon R, Serena C, Gene J, et al. In vitro antifungal susceptibili-
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8. Oliveira DC, Lopes PG, Spader TB, et al. Antifungal susceptibilities
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epidemic of sporotrichosis in cats: 347 cases (1998-2001). J Am
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12. Crothers SL, White SD, Ihrke PJ, et al. Sporotrichosis: a retrospec-
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Vet Dermatol. 2009;20:249-259.
13. Madrid IM, Mattei AS, Fernandes CG, et al. Epidemiological find-
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14. Schubach TM, de Oliveira Schubach A, dos Reis RS, et  al. Spo-
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out sporotrichosis in Rio de Janeiro, Brazil. Mycopathologia.
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15. Moriello KA, Franks P, Delany-Lewis D, et  al. Cutaneous-lym-
phatic and nasal sporotrichosis in a dog. J Am Anim Hosp Assoc.
1987;24:621-626.
16. Schubach TM, Schubach A, Okamoto T, et  al. Canine sporotri-
chosis in Rio de Janeiro, Brazil: clinical presentation, laboratory
diagnosis and therapeutic response in 44 cases (1998-2003). Med
Mycol. 2006;44:87-92.
17. Shany M. A mixed fungal infection in a dog: sporotrichosis and
cryptococcosis. Can Vet J. 2000;41:799-800.
18. Teixeira PA, de Castro RA, Nascimento RC, et  al. Cell surface
expression of adhesins for fibronectin correlates with virulence in
Sporothrix schenckii. Microbiology. 2009;155:3730-3738.
19. Leme LR, Schubach TM, Santos IB, et al. Mycological evaluation
of bronchoalveolar lavage in cats with respiratory signs from Rio
de Janeiro, Brazil. Mycoses. 2007;50:210-214.
20. Schubach TM, Schubach Ade O, Cuzzi-Maya T, et  al. Pathol-
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21. Schubach TM, Schubach A, Okamoto T, et  al. Haematogenous
spread of Sporothrix schenckii in cats with naturally acquired spo-
rotrichosis. J Small Anim Pract. 2003;44:395-398.
22. Goad DL, Goad ME. Osteoarticular sporotrichosis in a dog. J Am
Vet Med Assoc. 1986;189:1326-1328.
23. Reis EG, Gremiao ID, Kitada AA, et  al. Potassium iodide cap-
sule treatment of feline sporotrichosis. J Feline Med Surg.
2012;14:399-404.
24. Sykes JE, Torres SM, Armstrong PJ, et  al. Itraconazole for treat-
ment of sporotrichosis in a dog residing on a Christmas tree farm.
J Am Vet Med Assoc. 2001;218:1440-1443:1421.
25. Whittemore JC, Webb CB. Successful treatment of nasal sporotri-
chosis in a dog. Can Vet J. 2007;48:411-414.
26. Pereira SA, Menezes RC, Gremiao ID, et  al. Sensitivity of cyto-
pathological examination in the diagnosis of feline sporotrichosis.
J Feline Med Surg. 2011;13:220-223.
27. de Oliveira MM, Sampaio P, Almeida-Paes R, et al. Rapid identi-
fication of Sporothrix species by T3B fingerprinting. J Clin Micro-
biol. 2012;50:2159-2162.
28. Fernandes GF, Lopes-Bezerra LM, Bernardes-Engemann AR, et al.
Serodiagnosis of sporotrichosis infection in cats by enzyme-linked
immunosorbent assay using a specific antigen, SsCBF, and crude
exoantigens. Vet Microbiol. 2011;147:445-449.
29. Bernardes-Engemann AR, Costa RC, Miguens BR, et  al. Devel-
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30. Assi M, Lakkis IE, Wheat LJ. Cross-reactivity in the Histoplasma
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cine Immunol. 2011;18:1781-1782.
31. Hu S, Chung WH, Hung SI, et al. Detection of Sporothrix schenckii
in clinical samples by a nested PCR assay. J Clin Microbiol.
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32. Kano R, Watanabe K, Murakami M, et al. Molecular diagnosis of
feline sporotrichosis. Vet Rec. 2005;156:484-485.
33. de Miranda LH, Quintella LP, dos Santos IB, et al. Histopathology
of canine sporotrichosis: a morphological study of 86 cases from
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34. Miranda LH, Quintella LP, Menezes RC, et  al. Evaluation of
immunohistochemistry for the diagnosis of sporotrichosis in dogs.
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35. Vasquez-del-Mercado E, Arenas R, Padilla-Desgarenes C. Sporotri-
chosis. Clin Dermatol. 2012;30:437-443.
36. Burke MJ, Grauer GF, Macy DW. Successful treatment of cutaneo-
lymphatic sporotrichosis in a cat with ketoconazole and sodium
iodide. J Am Anim Hosp Assoc. 1983;19:542-547.
37. Anderson NV, Ivoghli D, Moore WS, et al. Cutaneous sporotricho-
sis in a cat: a case report. J Am Anim Hosp Assoc. 1973;9:526-529.
38. Heidrich D, Stopiglia CD, Senter L, et  al. Successful treatment
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2011;86:S182-S185.
39. Gremião I, Schubach T, Pereira S, et  al. Treatment of refractory
feline sporotrichosis with a combination of intralesional ampho-
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41. Carlos IZ, Sassa MF, da Graca Sgarbi DB, et al. Current research
on the immune response to experimental sporotrichosis. Myco-
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sporotrichosis in Rio de Janeiro, Brazil: epidemiological aspects of
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CHAPTER 65
Aspergillosis
Jane E. Sykes
Overview of Aspergillosis
First Described: Invasive aspergillosis was first described in
1815 in a jackdaw bird (Mayer)1
Cause: Aspergillus spp. (an ascomycete, teleomorphs Neosar-
torya, Emericella, Eurotium)
Affected Hosts: Dogs, cats, humans, and a variety of other
animal species
Geographic Distribution: Worldwide
Mode of Transmission: Inhalation of conidia from the
environment
Major Clinical Signs: Sinonasal aspergillosis: nasal discharge,
sneezing, epistaxis, depigmentation of the nares in dogs.
Sino-orbital aspergillosis (especially cats): exophthalmos,
mass lesions or ulcers in the pterygopalatine fossa. Dis-
seminated aspergillosis: inappetence, weight loss, spinal
pain, lameness, pelvic limb paresis or paralysis, cough,
vomiting.
Differential Diagnoses: Sinonasal aspergillosis: neoplasia,
inflammatory rhinitis, oronasal fistulas, cryptococcosis,
nasal mites (dogs), foreign bodies, and polyps (cats). Dis-
seminated aspergillosis: bacterial discospondylitis, bacte-
rial pyelonephritis, and other systemic fungal infections,
especially paecilomycosis and penicilliosis.
Human Health Significance: Human Aspergillus infections are
acquired from the environment. Direct transmission from
affected dogs and cats to humans does not occur.
A
spergillosis is the disease caused by pathogenic fungi that
belong to the genus Aspergillus. Aspergillus species are
ubiquitous, saprophytic, hyaline molds, the spores of
which can be found in soil, water, air, and decaying vegetation.
It is estimated that an individual person inhales several hundred
Aspergillus spores each day.2 Aspergillus species cause a variety
of noninvasive, semi-invasive, and invasive forms of disease in
dogs and cats that include keratomycosis, fungal otitis externa,
sinonasal aspergillosis (SNA), sino-orbital aspergillosis (SOA),
bronchopulmonary aspergillosis, and disseminated aspergillosis.
Aspergillus species that can cause disease in dogs and cats are
shown in Table 65-1.3-14 Teleomorphs (sexual forms) of the fungus
have been identified for some Aspergillus species. Currently, these
are named differently and include fungi that belong to the genera
Neosartorya, Emericella, and Eurotium. The name Aspergillus
may be retained even when teleomorphs are isolated from lesions,
to avoid confusion through the use of unfamiliar names.12 Because
accurate identification of Aspergillus species (especially rare spe-
cies) requires advanced molecular techniques, organisms can be
misidentified based on their morphology in culture alone. As a
result, use of the term “species complex” is recommended when
molecular techniques are not used for identification purposes.15
Because the etiology, epidemiology, clinical signs, diagnosis,
and treatment of upper respiratory tract (URT) aspergillosis,
systemic aspergillosis, and keratomycosis/otomycosis differ,
they are considered separately in this chapter.
Upper Respiratory Tract Aspergillosis
Etiology and Epidemiology
In dogs, SNA is a subacute to chronic, noninvasive disease
of the nasal cavity and sinuses. It is most often caused by
­Aspergillus fumigatus. Rarely, infection by other species, such
as A­ spergillus niger complex organisms (such as Aspergillus
tubingensis) or Aspergillus flavus and the closely related fun-
gus Penicillium, occurs. SNA occurs worldwide. Any breed of
dog can be affected, but disease is most common in large, non-
brachycephalic breeds, especially German shepherd dogs, Rott-
weilers, Border collies, Rhodesian ridgebacks, and retrievers.
Dogs of any age can be affected (median age around 6 years).16
No clear sex predisposition exists. SNA is thought to occur
when local immune mechanisms fail as a result of uncharac-
terized genetic or acquired immunodeficiency syndromes, and
occasionally as a result of implantation of a foreign body or
other preexisting nasal disease, such as nasal neoplasia. Rarely,
it occurs in conjunction with other systemic diseases such as
hyperadrenocorticism, diabetes mellitus, or systemic autoim-
mune disorders. However, in most cases, affected dogs are
otherwise systemically well and have no evidence of immune
compromise. Impaired cell-mediated immune responses have
been detected in dogs with SNA, but it is unclear whether this is
a cause or consequence of Aspergillus infection.17
Upper respiratory tract aspergillosis is relatively uncommon in
cats, and includes SNA and SOA. Approximately 50 cases of URT
aspergillosis have been reported in cats to date.3 Fungi that belong
to the A. fumigatus complex and, rarely, other Aspergillus species
may also be involved (see Table 65-1). A novel organism with a
proposed name of Aspergillus felis sp. nov. appears to cause SOA
in cats,12 whereas SNA is caused predominantly by A. fumigatus
sensu stricto.3 Brachycephalic breeds such as Persians and Hima-
layan Persians appear to be predisposed to SNA and especially
to SOA, but both conditions occur in any breed of cat. Affected
cats range in age from 1 to 13 years (median, 5 years). No clear
sex predisposition exists.3 Most cats with URT aspergillosis test
negative for retrovirus infection; only one cat with SNA in the
633
634 SECTION 3  Fungal and Algal Diseases

TABLE 65-1
Aspergillus Species Associated with Disease in Dogs and Cats3-14
Clinical Form Host Species Aspergillus species*
Keratomycosis Cats A. fumigatus
A. flavus
Dogs Not reported
Otomycosis Cats A. fumigatus
Dogs (very rare) A. ochraceus
A. versicolor
A. niger
Sinonasal Dogs A. fumigatus
aspergillosis A. niger
A. nidulans
A. flavus
A. tubingensis
Cats A. fumigatus FIGURE 65-1  Unilateral depigmentation and ulceration of the nares of a 7-year-old
shepherd mix with sinonasal aspergillosis. (Courtesy Dr. Lynelle Johnson, Small Animal
A. niger
Internal Medicine Service, University of California, Davis.)
A. udagawae
A. flavus
Neosartorya spp. ciliostasis and apoptosis and can suppress the host immune
Sino-orbital Cats A. felis sp. nov. response.2 The fungus also produces proteases and phospholi-
aspergillosis pases. Chemokines such as Il-8 and monocyte chemoattractant
protein-1 (MCP-1/CCL2) are secreted by host inflammatory cells
Bronchopulmonary Dogs and cats A. fumigatus in response to the fungus, which may recruit neutrophils and mac-
aspergillosis A. flavus (dog) rophages to the site of infection.20 Severe disease is associated with
Disseminated Dogs A. terreus destruction of nasal bones, the cribriform plate, and/or the orbit.
aspergillosis A. deflectus Clinical signs of SNA in dogs and cats consist of sneezing,
A. fumigatus reverse sneezing, serous to mucopurulent nasal discharge, depig-
A. niger mentation of the nasal planum, and, less commonly, epistaxis.
A. flavipes Nasal discharge is initially unilateral but can become bilateral as
A. lentulus disease progresses. Animals with advanced disease can be lethar-
A. carneus gic and inappetent and may lose weight. Nasal pain can mani-
A. versicolor fest in dogs as pawing at the face, hiding, or withdrawal when
A. alabamensis approached. Destruction of the nasolacrimal duct or invasion of
A. felis sp. nov. the orbit can lead to epiphora. Osteolysis of the cribriform plate
Cats A. fumigatus is often subclinical, but occasionally leads to meningitis with
neurologic signs such as obtundation or seizures.
*Some isolates have not been identified beyond the species complex In cats with SOA, Aspergillus invades the submucosal tissue
level (including some A. flavus, A. fumigatus, and A. niger). of the nasal cavity and sinus and extends through the orbital
bone and into the retrobulbar space. A profound granulomatous
inflammatory response to the fungus results in the formation of
literature was co-infected with FeLV.18 Five of 8 cats with SNA a retrobulbar mass, with clinical signs that include exophthal-
evaluated at the author’s teaching hospital had diabetes mellitus.4 mos, protrusion of the third eyelid, mass lesions in or ulceration
of the pterygopalatine fossa, and, rarely, blindness due to inva-
Clinical Features sion of the optic nerve or optic chiasm.3,21-23 Inappetence and
dysphagia can also develop.22 Occasionally, the clinical signs
Signs and Their Pathogenesis of SOA develop after initial signs of SNA, such as sneezing and
Aspergillus conidia (or spores), which are 2 to 3 µm in diameter, nasal discharge.3,21
are inhaled and deposit in the nasal cavity and/or sinuses, where
they adhere to epithelial cells. In the absence of adequate immune Physical Examination Findings
defenses, the conidia enlarge and germinate to form hyphae. SNA Physical examination findings in dogs with SNA include unilat-
is a noninvasive disease, and the fungus does not extend beyond eral or bilateral serous or mucopurulent nasal discharge, epistaxis,
the mucosal epithelium.19 Production of toxins by the fungus and pain on palpation of the face, mild mandibular lymphadenomeg-
a profound host inflammatory response is thought to explain aly, and sometimes depigmentation and ulceration of the nares
the extensive turbinate and bony destruction that follows fungal (Figure 65-1).24,25 Nasal airflow is usually normal or increased
colonization. Toxins produced by A. fumigatus include aflatoxin, because of turbinate lysis. Serous ocular discharge occurs uncom-
hemolysin, fumagillin, and gliotoxin,2 but the role that these tox- monly as a result of destruction of the bones of the orbit. Rarely,
ins play in canine SNA has not been determined. Gliotoxin induces palpation of the nasal bones or the hard palate can reveal defects
 CHAPTER 65  Aspergillosis 635

A B

C D
FIGURE 65-2  A, Seven-year-old male neutered Scottish fold cat with exophthalmos, conjunctivitis, and blindness of the right eye secondary to sino-orbital aspergillosis. A 1- to 2-cm
mass was also identified in the pterygopalatine fossa on oral examination. B-D, MRI image of the head of the same cat. The right eye was displaced rostrally by a large retrobulbar mass that
is mildly, heterogeneously hyperintense on T1-weighted images (B), hyperintense on T2-weighted images (C), and enhanced with contrast (D). The mass extends medial to the ramus of
the mandible and enters the nasopharynx as well as the oropharynx. A small T1-hyperintense structure is noted in the left frontal sinus.

in the underlying bone. Dogs with cribriform plate involvement Diagnosis

and meningitis may be obtunded or dehydrated. Dogs with severe
epistaxis can have evidence of melena on rectal examination sec-
ondary to swallowed blood. Most affected dogs are afebrile.
Physical examination findings in cats with SNA consist of
serous or mucopurulent nasal discharge, sneezing, stertor, epi-
staxis, and, in some cases, facial distortion.3 Cats with SOA
usually have unilateral exophthalmos and deviation of the
globe; bilateral involvement has also been described (Figure
65-2).3,22,23 There may be resistance to ocular retropulsion and
protrusion of the third eyelid. Miosis, an absent pupillary light
response, and an absent menace response may be present. On
examination of the oral cavity, most affected cats also have
a mass or ulcer in the pterygopalatine fossa ipsilateral to the
affected eye. Other less common findings are exposure keratitis
and corneal ulceration, mandibular lymphadenomegaly, pain
on opening the mouth, and/or ulceration of the hard palate.3,22
Early diagnosis of URT aspergillosis relies on a high degree
of suspicion for the disease and availability of client finan-
cial resources, because there is a lack of sensitive, noninvasive
diagnostic tests for the disease. Definitive diagnosis requires
a combination of imaging, visualization of fungal plaques by
rhinoscopy/sinuscopy, and identification of fungal structures
on cytology or histopathology. Imaging should be performed
before rhinoscopy to avoid the influence of iatrogenic hemor-
rhage on the results.
Laboratory Abnormalities
Dogs with SNA often have no abnormalities on the CBC, serum
chemistry profile, or urinalysis. Occasionally, mild nonregen-
erative anemia; mild neutrophilia, rarely with a left shift; mild
eosinophilia; and/or mild hypoalbuminemia are present.4 Cats
636 SECTION 3  Fungal and Algal Diseases

A B

C D
FIGURE 65-3  Plain radiographs (A and B) and CT scan images (C and D) of the nasal cavity of a 6-year-old female spayed golden retriever with sinonasal aspergillosis. A, Open-mouth
view. There is marked loss of detail in the left nasal cavity. B, Skyline projection of the frontal sinuses. There is increased opacity in the left frontal sinus. C, A transverse image through the
nasal cavity shows marked turbinate loss on the left side. D, Transverse image through the frontal sinuses and region of the cribriform plate. There is moderate fluid accumulation and irregu-
lar soft tissue structures in the left frontal sinus, and the frontal bone that overlies the sinus is thickened and irregular. Bony destruction at the level of the cribriform plate is present (arrow).

with SOA may have a mild mature neutrophilia and sometimes
mild to severe hyperglobulinemia.3,21
Diagnostic Imaging
Advanced Imaging
Whenever possible, computed tomography (CT) or MRI are pre-
ferred imaging modalities for the nasal cavity because of their
increased sensitivity over plain radiography for evaluation of the
nasal cavity, sinuses, and cribriform plate.26 Bony changes are
best appreciated on CT. Findings on CT or MRI in canine and
feline SNA include loss of nasal turbinates and defects in the
nasal septum; increased soft tissue opacity within the nasal cavity
and/or sinuses due to mucosal inflammation, fluid accumulation,
or fungal plaques; hyperostosis of the frontal bones; and man-
dibular or retropharyngeal lymph node enlargement.3,26-29 Oste-
olysis of the cribriform plate, orbit, frontal bones, and, rarely,
the maxillary or palatine bones may be present (Figure 65-3, C).
Meningeal enhancement is sometimes evident on contrast

FIGURE 65-4  Rhinoscopic view of a large gray fungal plaque in the nasal cavity of an
8-year-old female pit bull terrier with sinonasal aspergillosis.
administration in dogs with cribriform plate destruction.25 Fun-
gal plaques and mucosal secretions fail to enhance with contrast
and cannot be readily distinguished from each other. In some
dogs, lesions are limited to the frontal sinus.27
Advanced imaging of the head in cats with SOA reveals a
retrobulbar mass that enhances diffusely or heterogeneously
with contrast. Displacement of the globe, osteolysis of the orbit,
mandibular lymphadenomegaly, and changes consistent with
SNA can also be present. With MRI, retrobulbar masses are
T2-hyperintense. Invasion of temporal and masseter muscles by
the mass may also be apparent on MRI (see Figure 65-2).30
Plain Radiography
Plain skull radiographs in dogs with SNA may show unilateral
or bilateral loss of turbinate detail, destruction of the nasal sep-
tum, and/or increased soft tissue opacity within the nasal cav-
ity. The open-mouth view provides optimum evaluation of the
turbinates (see Figure 65-3, A). A “skyline” view of the nasal
sinuses may show opacification of one or both sinuses and fron-
tal bone hyperostosis (see Figure 65-3, B).26
Rhinoscopy and Sinuscopy
Visualization of fungal plaques by rhinoscopy and sinuscopy con-
firms a diagnosis of SNA. Plaques are white, gray, yellow, black,
greenish, or a mixture of these colors and are adhered to the nasal
mucosa or mucosa of the frontal sinus (Figure 65-4). Caudal rhi-
noscopy is performed with a flexible endoscope, after which a
rigid telescope or flexible endoscope is used to examine the ros-
tral portions of the nasal cavity. In some cases, extensive turbi-
nate destruction allows a flexible endoscope to be passed directly
into the frontal sinuses through the nasofrontal opening. If this
is not possible, but CT or MRI findings suggest the possibility of
frontal sinus lesions, trephination or surgical exploration of the
frontal sinuses is necessary to visualize (and treat) this region.
After trephination, a rigid endoscope can be used to examine the
frontal sinus through the trephination site.
CHAPTER 65  Aspergillosis 637
Microbiologic Tests
Cytologic Examination
Cytologic examination of blind or rhinoscopy-directed mucosal
swabs, brush specimens of the nasal cavity, or nasal biopsies
from dogs or cats with SNA can reveal Aspergillus hyphae and
sometimes also conidia. Hyphae stain well with Wright’s stains.
They are septate and branch at 45-degree angles (see Figure
4-3). Large numbers of degenerate neutrophils, ciliated epithe-
lial cells, and/or bacteria are also usually present. In one study,
the sensitivity of cytologic examination was 93% when cytol-
ogy brush specimens were examined and 100% when squash
preparations of biopsies were examined, but it was 20% or less
when nasal discharge or smears made from blind swab speci-
mens were examined.31
Cytologic examination of ultrasound- or CT-guided aspi-
rates of retrobulbar masses from cats with SOA reveals a mixed
inflammatory response and sometimes fungal hyphae.
Fungal Culture
Aspergillus fumigatus can be readily grown on routine labora-
tory media. Colonies of mold generally appear within 96 hours,
but occasionally longer incubation times are required.32 In the
absence of supportive rhinoscopic, cytologic, or histopathologic
findings, positive cultures from the nasal cavity (or other non-
sterile sites) require cautious interpretation because Aspergillus
may be found within the URT of healthy animals. Nevertheless,
in two separate studies, no dogs with nonfungal nasal disor-
ders had positive culture results for A ­ spergillus.16,32 In one of
these studies (which included 21 dogs with SNA and 37 dogs
with nonfungal nasal disease), the sensitivity of fungal culture
of nasal biopsies for diagnosis of canine SNA was 81%.16 In
the other study, fungal culture of biopsy specimens or plaque
specimens collected by rhinoscopic guidance (≥75%) was more
sensitive than culture of blindly collected nasal swab specimens
(<20%).32
Growth of an A. fumigatus complex organism from guided
aspirates or biopsy specimens collected from a retrobulbar mass
in a cat strongly suggests a diagnosis of SOA. In one study, fun-
gal culture of nasal biopsies or aspirates from cats with SOA
and SNA had a sensitivity greater than 95%.3
Serologic Diagnosis
Both serum antibody and antigen assays have been evaluated
for diagnosis of URT aspergillosis. Assays for detection of
serum antibodies to Aspergillus include ELISA and gel immuno-
diffusion (ID) assays (see Figure 2-7). Assay types vary in their
sensitivity and specificity for diagnosis of SNA, and clinical per-
formance data for some assays used in commercial veterinary
diagnostic laboratories may not be available. The sensitivity of
one gel ID assay (Meridian Bioscience) for diagnosis of canine
SNA was 57% to 77%,16,25,33 and the specificity was at least
98%.16,33 The sensitivity and specificity of another gel ID assay
(Immuno-Mycologics, OK) was 31% and 97%, respectively.34
The sensitivity and specificity of an ELISA that detects antibod-
ies against a purified Aspergillus antigen extract was 88% and
97%, respectively.33 Thus, negative results with these antibody
assays do not rule out SNA, but positive results strongly suggest
the disease is present. The sensitivity and specificity of serum
antibody assays for diagnosis of feline URT aspergillosis has not
been evaluated in a large number of cats, but both positive and
negative assay results have been reported in affected cats.3,28,35
638 SECTION 3  Fungal and Algal Diseases
A B
FIGURE 65-5  Histopathology of a nasal biopsy from a 4-year-old intact male Border collie with sinonasal aspergillosis. A, There is a profound neutrophilic inflammatory response
beneath a thick mat of fungal hyphae, which stain magenta. Periodic acid–Schiff stain, 400× magnification. B, Aspergillus hyphae (arrowheads), conidiophores (small arrows) and conidia
(spores) can be seen on the surface of the lesion. H&E stain, 1000× oil magnification.
A serum Aspergillus galactomannan antigen ELISA assay
(Platelia), originally developed for diagnosis of invasive asper-
gillosis in humans, has been studied for the diagnosis of SNA
and SOA in dogs and cats, but its sensitivity and specificity have
been low.5,33,36,37 Only 4 of 17 dogs with SNA were positive in
one study, and only weak positive results were obtained (galac-
tomannan indices 0.5-0.8, reference range <0.5).33 False positive
results occurred in 11 of 62 dogs without SNA (galactomannan
indices of 0.5-1.5). Only 3 of 13 cats with URT aspergillosis
tested positive, and false-positive results were obtained in a
number of cats without URT aspergillosis (specificity 78%).38
Molecular Detection Using the Polymerase Chain Reaction
The use of real-time PCR assays that detect Aspergillus DNA in
nasal biopsy specimens has been evaluated for diagnosis of SNA
in dogs, but assays evaluated had either unacceptably high rates
of false-positive test results (PenAsp assay) or a low sensitivity
(Aspergillus genus-specific PCR).34 For both assays, dogs with
SNA had higher DNA copy numbers than control dogs or dogs
with lymphoplasmacytic rhinitis or neoplasia, but there was
considerable overlap in copy numbers between dogs with SNA
and those without SNA.
Pathologic Findings
Histopathology of nasal biopsies from dogs with SNA usually
reveals branching, septate fungal hyphae in a background of lym-
phoplasmacytic and neutrophilic inflammation; occasionally the
neutrophilic component predominates. Variable numbers of mac-
rophages and eosinophils may also be present, as well as necrotic
debris. Other findings include osteolysis, turbinate remodeling,
and intralesional bacteria. Occasionally, intralesional plant mate-
rial is detected. Mats of fungal hyphae may be present if a plaque
is submitted for examination. Conidiophores and conidia can be
found because the fungus is exposed to air within the nasal cav-
ity (Figure 65-5). The sensitivity of rhinoscopically guided nasal
biopsy for diagnosis of canine SNA in one study was 82% (18
of 22 dogs).25 In that study, three to five biopsy specimens were
collected from each side of the nasal cavity.
Histopathology of biopsies of retrobulbar mass lesions in cats
with SOA reveals granulomatous inflammation with necrosis and
intralesional branching hyphae. A mixed inflammatory reaction
(epithelioid macrophages, eosinophils, neutrophils, lymphocytes,
and plasma cells) and a peripheral zone of fibrosis surround the
granulomas. Inflammatory lesions may efface muscle and bone.
Treatment and Prognosis
Successful treatment of SNA is challenging. Because of the large size
of the nasal cavity and sinuses, clinical signs are often not apparent
until disease is advanced. Delays in diagnosis can occur because
of the costly and invasive nature of diagnosis and treatment and
the need for specialized imaging techniques and endoscopic exper-
tise. Owners of adult dogs that develop persistent nasal discharge
(e.g., present for at least 1 week) should be encouraged to pursue
advanced imaging and rhinoscopy as soon as possible so that the
disease can be recognized and treated early. Treatment with anti-
bacterial drugs alone is discouraged, because primary bacterial
infections of the nasal cavity in adult dogs are rare.
In general, treatment of SNA requires thorough mechanical
debridement of plaques with endoscopic biopsy forceps until no
visible fungus remains, which may necessitate extended anes-
thesia. In some cases, treatment is performed immediately after
imaging and rhinoscopy, which further prolongs the period of
anesthesia. If plaques within the frontal sinuses cannot be accessed
by antegrade rhinoscopy through the nasofrontal opening, fron-
tal sinus trephination and curettage or open surgical debridement
of the frontal sinus may be required. Provided the cribriform
plate is intact, debridement is followed by repeated lavage with
0.9% saline and suction, followed by topical antifungal drug
therapy with clotrimazole or enilconazole. Additional systemic
antifungal drug treatment could also be considered, but whether
this improves outcome over topical therapy alone is unknown.
Topical Antifungal Drug Treatment
A variety of noninvasive and invasive protocols for topical anti-
fungal drug treatment of canine SNA have been described with
varied rates of success. Currently, there is a lack of masked,
randomized clinical trials with consistent and long-term follow-
up evaluation that indicate clear benefit of one treatment over
another. The protocol described here is used at the author’s
institution. A similar protocol has been used successfully to
treat SNA in cats.38

Cribriform
plate
Infusion
catheter
Sponge
Foley
catheter
Endotracheal Soft palate
tube
FIGURE 65-6  Diagram illustrating placement of catheters for topical treatment of
canine sinonasal aspergillosis with clotrimazole or enilconazole.
A 24F Foley catheter is retroflexed over the soft palate and
the balloon inflated until it can be palpated above the soft palate
(Figure 65-6). Placement of the catheter is facilitated by inser-
tion of a stiff guidewire into the catheter. The tip of the cath-
eter can then be bent into a U shape and hooked over the soft
palate, after which the balloon is inflated. The nasopharynx is
packed with moistened laparotomy pads, a gauze pad is placed
over the incisive papilla, and the proximal end of the catheter
is clamped across the guidewire to prevent leakage of antifun-
gal drug from the nasal cavity. A 10F polypropylene catheter
is inserted into each nostril to the level of the medial canthus,
or alternatively these two catheters can be placed directly into
the frontal sinus with endoscopic guidance.24,39 The tip of an
additional 12F Foley catheter is inserted into each of the nares,
the balloon inflated, and the catheters clamped so the nares
are occluded. Cotton-tipped swabs can also be inserted into
the nares to prevent outflow of antifungal drug. Clotrimazole
(1%) or enilconazole (1% or 2%) solution is then administered
through the polypropylene catheters. For large-breed dogs, 50 to
60 mL of solution is administered per side, and if frontal sinus
trephination was performed, half of the solution can be adminis-
tered into a catheter placed in the trephination site. The patient
is rotated 45 degrees every 15 minutes over an hour to distribute
the drug into the nasal cavity and sinuses. The catheters and
sponges are then removed with the dog’s head tilted downward
at an angle of 30 degrees, and the material is allowed to drain
from the nasal cavity for 10 minutes. Debridement and a second
treatment are then routinely performed 1 month later; some-
times, a third treatment may be necessary. Clotrimazole can be
irritating to tissues, and solutions that contain propylene glycol
can cause pharyngeal irritation and edema.40 Enilconazole solu-
tions may be less irritating, but there is no evidence that they are
more effective than clotrimazole.
When cribriform plate destruction is present, topical treatment
can lead to the development of life-threatening neurologic signs or
failure to recover from anesthesia. Nevertheless, treatment has also
been performed without complication in some dogs with minor
defects in the cribriform plate.25 Systemic azole antifungal drug
therapy could be considered as an alternative in this situation.
Use of topical antifungal creams has been studied in order
to improve retention in the sinonasal cavities and reduce proce-
dure time.24,41 In one study, 1% clotrimazole solution (25 or 60
mL based on patient size) was administered through a catheter
placed through a trephination site in each frontal sinus, followed
by a 1% clotrimazole cream (10 or 20 g).41 Debridement was
not performed, so the mean treatment time was only 32 minutes.
CHAPTER 65  Aspergillosis 639
Of 14 dogs treated, 10 had resolution of clinical signs, but out-
come was based only on telephone follow-up at 6 to 24 months,
and not endoscopic reevaluation. In another study, 15 g of 1%
bifonazole cream was administered into the frontal sinus using
endoscopic guidance either alone or after topical treatment with
enilconazole.24 Dogs were also treated with ketoconazole or
itraconazole. All 12 dogs treated with a combined enilconazole-
bifonazole treatment were considered cured after a second treat-
ment, but 2 of 5 dogs treated with bifonazole alone had persistent
disease. Bifonazole has limited availability in the United States.
Systemic Antifungal Drug Treatment
Although their efficacy for treatment of SNA has been debated,
azole antifungals with activity against molds (i.e., ketoconazole,
itraconazole, posaconazole, or voriconazole) have been used
either as monotherapy for dogs with cribriform plate involvement
or in combination with topical antifungal drugs. Debridement
before commencing systemic antifungal drug treatment is recom-
mended if client finances permit, because effective drug concen-
trations are not likely to be obtained within fungal plaques.
Cats with SOA must be treated with systemic antifungal
drugs such as an azole (itraconazole or posaconazole) and/or
amphotericin B; the use of terbinafine in combination with these
drugs has also been reported.3 One cat was successfully treated
with caspofungin followed by posaconazole monotherapy.
More than 50% of treated cats failed treatment in one study.3
The optimum treatment for SOA requires further study.
Prognosis
The prognosis for SNA depends on the extent of disease at the
time of diagnosis. Success rates vary from less than 30% to
100%, but methods to assess treatment success and follow-up
times have not been consistent. Serial serologic testing by gel ID
is not useful to monitor treatment success.25 Even if infection is
eliminated based on endoscopic reevaluation and biopsy, dogs
with extensive turbinate loss are predisposed to recurrent bacte-
rial infections of the nasal cavity, which result in persistent nasal
discharge and sneezing. One study showed no difference in the
prevalence of sneezing and nasal discharge between dogs that
responded to initial treatment and dogs that failed to respond.25
Recurrence of SNA due to persistent infection or re-infection
by Aspergillus spp. can also occur. The prognosis for complete
remission of SOA in cats is guarded to poor.
Public Health Aspects
There are no reports of transmission of SNA from affected dogs
to humans. Whether the handling of affected dogs by immuno-
suppressed humans poses a risk to their health is unknown, but
such individuals should probably minimize contact with affected
animals. If contact is necessary, they should be encouraged to
discuss the problem with their medical practitioner. At the least,
gloves should be worn and proper hand washing practiced.
Systemic Aspergillosis
Etiology and Epidemiology
Systemic aspergillosis may involve the respiratory tract (bron-
chopulmonary aspergillosis) or may be a disseminated disease of
multiple organ systems. Localized bronchopulmonary aspergil-
losis is rare in dogs and cats, and often follows underlying local
640 SECTION 3  Fungal and Algal Diseases

or systemic immunodeficiency.6,42-44 German shepherds appear

to be predisposed to a form of bronchopulmonary aspergillosis TABLE 65-2
characterized by the development of cavitary lung lesions.6,45-47 Historical Signs in 30 Dogs with Disseminated Aspergillosis
Bronchopulmonary aspergillosis is typically associated with
infection by A. fumigatus complex fungi, whereas disseminated Sign Percent of Dogs
aspergillosis in dogs is most often caused by Aspergillus terreus
or Aspergillus deflectus. Other Aspergillus species have also been Inappetence or anorexia 20
isolated from dogs with disseminated disease (see Table 65-1). Pain 27
In dogs, disseminated aspergillosis may occur as a result of Lameness 13
an uncharacterized genetic immunodeficiency, because female
Neurologic signs (especially pelvic 20
German shepherds are strongly predisposed to the disease,
limb ataxia and paresis)
and immunosuppressive drug treatment is rarely present in the
history of affected dogs.7 Two thirds of affected dogs seen at Respiratory signs (especially cough) 13
the author’s institution were German shepherds, and German Vomiting  7
shepherds were 43-fold more likely to develop the disease than
other dog breeds.7 Rhodesian ridgebacks were also predisposed. From Schultz RM, Johnson EG, Wisner ER, et al. Clinicopathologic and
Females are 3 times as likely to develop disseminated aspergil- diagnostic imaging characteristics of systemic aspergillosis in 30 dogs.
J Vet Intern Med. 2008;22:851-859.
losis as males, and the median age of affected dogs is 4.5 years
(range, 2 to 8 years). Concurrent or sequential infections with
other opportunistic pathogens have been described in some
dogs.4,48
Disseminated and bronchopulmonary aspergillosis are rare TABLE 65-3
in cats.42,49,50 Affected cats often have recognizable underlying
immunosuppressive conditions such as diabetes mellitus, cancer
Physical Examination Findings in 30 Dogs with Disseminated
chemotherapy, or severe viral infections. The remainder of this Aspergillosis
chapter focuses on systemic aspergillosis in dogs.
Finding Percent of Dogs
Clinical Features Muscle wasting/thin body condition 40
Fever 27
Signs and Their Pathogenesis
Spinal pain 17
Pulmonary or disseminated aspergillosis is thought to develop
after inhalation of Aspergillus conidia, which lodge in the alve- Vestibular signs 17
oli and adhere to epithelial cells. In the absence of adequate Peripheral lymphadenomegaly 17
local immune defenses, the conidia enlarge and germinate to Ocular abnormalities 13
form hyphae. Invasion of the bloodstream and dissemination to
distant sites may occur as a result of systemic immunosuppres- Lameness 13
sion. Fungal virulence factors are also likely to be important, Ataxia 10
although why A. terreus and A. deflectus are more likely than Paraparesis 10
A. fumigatus to cause disseminated disease in dogs is unclear. Cough, increased breath sounds 10
Human disseminated aspergillosis is most often caused by
A. fumigatus–complex fungi.51 Mental dullness  7
Anatomic sites of predilection in dogs with disseminated Vision impairment  7
aspergillosis are the vertebral endplates and discs, renal pelvis, Hemiparesis  7
spleen, long bones, and lymph nodes. However, any organ may
Circling, seizures, arrhythmia  3
be affected, including the liver, heart, cardiac valves, pancreas,
eyes, lungs, brain, bone marrow, meninges, small intestine, From Schultz RM, Johnson EG, Wisner ER, et al. Clinicopathologic and
skin, spinal cord, thyroid, adrenal glands, prostate, and trachea. diagnostic imaging characteristics of systemic aspergillosis in 30 dogs.
Nasal cavity involvement is not generally a feature of the bron- J Vet Intern Med. 2008;22:851-859.
chopulmonary or disseminated forms, and dogs with dissemi-
nated disease often lack evidence of pulmonary involvement.
The median duration of illness before admission in one study with disseminated aspergillosis often have muscle wasting and/
was 1 month (range, 2 days to 9 months).7 The most frequent or a thin body condition. Fever (up to 104.2°F or 40.1°C) is
clinical signs are lethargy, inappetence, pain, lameness, and present in one quarter of affected dogs.7 Musculoskeletal abnor-
neurologic signs secondary to discospondylitis (especially ataxia malities include spinal pain, lameness, and, rarely, inability to
and paresis) (Table 65-2). Other signs include apparent blind- stand. Neurologic signs include vestibular abnormalities, ataxia,
ness, circling, head tilt, seizures, respiratory distress, and vomit- mental dullness, paraparesis, vision impairment, hemiparesis,
ing. Cough, increased respiratory effort, and rarely hemoptysis circling, and seizures. Dogs with respiratory involvement may
may occur in dogs with bronchopulmonary aspergillosis.6,47 cough and have harsh lung sounds and/or increased respiratory
effort. Peripheral lymphadenomegaly may be detected. Ocular
Physical Examination Findings abnormalities include chorioretinitis, hyphema, and panophthal-
Physical examination findings in dogs with disseminated asper- mitis.7 Rarely, arrhythmias are detected secondary to myocardial
gillosis are listed in Table 65-3. On physical examination, dogs involvement, but myocardial involvement is often subclinical.
 CHAPTER 65  Aspergillosis 641

TABLE 65-4
Selected Hematologic and Serum Biochemistry Analysis Results in 30 Dogs with Disseminated Aspergillosis
Median
Variable (Range) No. (%) with Low Values No. (%) with High Values Reference Range
Hematocrit (%) 42% 8 (31%) 0 40%-55%
(14%-46%)
Neutrophils 12,643 0 21 (81%) 3000-10,500
(cells/µL) (7584-28,321)
Band neutrophils 0 0 10 (38%) Rare
(cells/µL) (0-3051)
Monocytes 1608 0 16 (62%) 150-1200
(cells/µL) (499-3760)
Lymphocytes 1566 4 (15%) 0 1000-4000
(cells/µL) (115-3998)
Platelets 262,000 1 (4%) 0 150,000-400,000
(cells/µL) (86,000-400,000)
Albumin (g/dL) 2.7 6 (24%) 0 2.9-4.2
(1.3-3.6)
Globulin (g/dL) 4.4 0 12 (48%) 2.3-4.4
(2.3-5.8)
Creatinine (g/dL) 1.1 0 9 (36%) 0.5-1.6
(0.5-7.8)
BUN (mg/dL) 19 0 9 (36%) 8-31
(8-196)
Calcium (mg/dL) 10.8 0 8 (32%) 9.9-11.4
(9.9-13.9)
ALT (IU/L) 37 0 3 (12%) 19-67
(19-609)
ALP (IU/L) 78 0 3 (12%) 15-127
(21-1920)

Diagnosis Cerebrospinal Fluid Analysis

Laboratory Abnormalities
Complete Blood Count
Most dogs with disseminated aspergillosis have leukocyto-
sis due to a neutrophilia (Table 65-4).7 A left shift and toxic
neutrophils may also be present. Normocytic, normochromic
nonregenerative anemia occurs in approximately one third of
affected dogs and may be secondary to inflammatory disease or
renal failure.
Serum Biochemical Tests
Common abnormalities in the serum biochemical profiles of
dogs with systemic aspergillosis include hyperglobulinemia, azo-
temia, hypercalcemia, and hypoalbuminemia (see Table 65-4).
Urinalysis
The urine specific gravity of azotemic dogs with disseminated
aspergillosis is usually in the isosthenuric range because of
renal failure. Hematuria, proteinuria, and pyuria may also be
detected. Fungal hyphae were identified in the urine sediment
of 2 (8%) of 25 dogs with disseminated aspergillosis in one
study.7
Analysis of the CSF in dogs with neurologic signs or spinal pain
due to disseminated aspergillosis can show no abnormalities,
or an increased nucleated cell count and CSF protein concen-
tration. The differential cell count reveals mixed or primarily
mononuclear or neutrophilic pleocytosis. Abnormalities of the
CSF were identified in 8 of 10 dogs with disseminated aspergil-
losis at the author’s teaching hospital.4
Diagnostic Imaging
Plain Radiography
Plain thoracic radiographs in dogs with bronchopulmonary
aspergillosis may show a bronchial pattern or lung lobe consoli-
dation.44 Consolidation and cavitary lesions may be seen in the
lungs of German shepherd dogs (Figure 65-7).45-47 Abnormali-
ties in dogs with disseminated aspergillosis include enlarged tra-
cheobronchial and/or sternal lymph nodes, cranial mediastinal
masses, pleural effusion, and/or pulmonary alveolar infiltrates.7
Spinal radiographs reveals evidence of discospondylitis in
approximately half of dogs with disseminated aspergillosis, with
a median of 2 sites affected (range, 1 to 9 sites).7 Productive and
destructive bony changes consistent with osteomyelitis may be
present in other parts of the skeleton (Figure 65-8). Most dogs
642 SECTION 3  Fungal and Algal Diseases

A B
FIGURE 65-7  Lateral thoracic radiograph of a 2-year-old male neutered German shepherd that had a 4-month history of cough and exercise intolerance. A, A cavitary lesion (arrows)
and alveolar infiltrates in the right middle lung lobe are present. B, On a horizontal beam projection, a fluid line is present (arrow) within the dependent aspect of the lesion.

FIGURE 65-9  Ultrasound image of the right kidney of a 6-year-old female spayed
German shepherd dog with disseminated aspergillosis. There is dilation of the renal pelvis,
which contains echogenic material (arrow).

abdominal lymph nodes.7 Renal abnormalities include pyelecta-

FIGURE 65-8  Lateral radiograph of the left humerus of a 2-year-old male neutered
German shepherd with disseminated aspergillosis. A primarily osteoproductive lesion is
present in the mid-diaphysis.
have multiple affected sites, which include the vertebral bodies,
sternebrae, ribs, scapula, humerus, and tibia.7
Sonographic Findings
Abdominal ultrasound abnormalities in dogs with dissemi-
nated aspergillosis are most often in the kidneys, spleen, and
sia, a distorted and mottled architecture, decreased corticome-
dullary distinction, echogenic debris within the renal pelvis and
proximal ureter, nodules or masses within the renal cortex, and
papillary blunting (Figure 65-9). Occasionally, hydronephrosis
is detected. Splenic abnormalities include hypoechoic nodules or
masses, splenomegaly, changes consistent with infarction, or a
mottled echotexture. Abdominal lymph nodes may be enlarged
and/or hypoechoic. Other findings include diffuse hepatic or
pancreatic hypoechogenicity, ascites, or evidence of venous
thrombosis.
Advanced Imaging
MRI of the head in dogs with Aspergillus infections that have
disseminated to the central nervous system (CNS) may reveal
multifocal lesions that are hypo- to isointense and enhance with
contrast on T1-weighted images. These lesions are hyperintense
or heterogenous on T2-weighted and fluid attenuated inversion
recovery (FLAIR) images. Mild or severe meningeal enhance-
ment may also be present.
 CHAPTER 65  Aspergillosis 643

TABLE 65-5
Diagnostic Assays Available for Aspergillosis in Dogs or Cats
Assay Specimen Type Target Performance
Cytologic Aspirates of affected Fungal hyphae False negatives can occur when specimen size is small or low
­examination tissues, body fluids, (with or without numbers of fungi are present. Cytology of brush specimens
impression smears of conidia in tissues optimizes sensitivity for dogs with SNA. ­Identification to
biopsies exposed to air) species level requires culture and ­molecular testing.
Fungal culture Aspirates or biopsies of Aspergillus species Sensitivity for diagnosis of canine SNA is at least 75% when
­affected tissues, body endoscopically guided nasal biopsies are cultured. Specific-
fluids ity is >95%. Culture of a typical species
(A. terreus or A. deflectus) from a dog with signs ­consistent
with disseminated disease strongly suggests disseminated
aspergillosis. Molecular testing by PCR may be required to
identify beyond the species complex level.
Antibody Serum Antibodies to Assays vary in sensitivity and specificity. In general, positive
­serology (gel ­Aspergillus species results support a diagnosis of canine SNA, but negative
ID or ELISA) results do not rule out the disease. Insensitive for diagnosis
of disseminated aspergillosis.
Galactomannan Urine, serum Aspergillus spp. Insensitive for diagnosis of SNA, SOA, and probably
antigen assay ­galactomannan bronchopulmonary disease. Very sensitive for diagnosis
(ELISA) cell wall antigen of disseminated aspergillosis. Cross-reactivity with other
fungal pathogens may occur (especially other molds). False
positives also occur with Plasmalyte administration.
Real-time PCR Whole blood Aspergillus spp. Assays studied lack sensitivity or specificity for diagnosis
assays DNA of canine SNA. Usefulness for diagnosis of disseminated
aspergillosis requires further study.
Histopathology Biopsy specimens from Fungal hyphae Sensitivity for diagnosis of canine SNA in rhinoscopically
­affected tissues (with or without guided biopsies was 82%. Hyphae are usually visualized in
conidia in tissues biopsies from animals with disseminated aspergillosis, but
exposed to air) lesions may be difficult to access for biopsy. Special stains
assist organism detection.

Microbiologic Tests cytologic evidence of fungal hyphae is required for definitive

Diagnostic assays available for aspergillosis in dogs or cats are
described in Table 65-5.
Cytologic Examination
Hyphae with morphology consistent with Aspergillus spp. may be
present in aspirates of lymph nodes, kidney, lung tissue, or bone
from dogs with systemic aspergillosis (see Figure 4-3).7 Cytologic
examination of pleural effusion, airway lavage specimens, or
urine may also reveal hyphae. False-negative results occur when
organism numbers are low or specimen size is small. Accurate
identification of the organism present requires fungal culture.
Fungal Culture
Aspergillus spp. are readily isolated on routine laboratory
media. Isolation of Aspergillus spp. (especially A. terreus or
A. deflectus) from normally sterile sites such as urine, blood,
or aspirates of lymph node, bone, intervertebral disc, liver, or
splenic tissue supports a diagnosis of disseminated aspergillo-
sis. Approximately 50% of dogs with disseminated aspergillosis
have positive urine cultures for Aspergillus spp., and around
one third of dogs have positive blood or CSF cultures.7 Isola-
tion of A. fumigatus from airway washes should be interpreted
more cautiously as it may represent contamination. Concurrent
diagnosis in this situation.
Once growth of mold occurs in culture, Aspergillus species
can be identified to the complex level based on conidiophore
morphology (Figure 65-10). Accurate identification to the
species level requires molecular techniques such as PCR, then
sequencing, which are performed by laboratories with special
expertise (e.g., the Fungus Testing Laboratory at the University
of Texas Health Science Center at San Antonio). Such labora-
tories can also perform antifungal drug susceptibility testing.
However, because clinical breakpoints have not been estab-
lished for dogs or cats, the relevance of minimum inhibitory
concentrations for molds remains unclear.
Serologic Diagnosis
An Aspergillus galactomannan antigen ELISA assay (Platelia)
has high sensitivity for diagnosis of disseminated aspergillosis in
dogs when serum or urine specimens are tested.36 In the author’s
hospital, all of 12 dogs with disseminated aspergillosis had posi-
tive test results, with galactomannan indices (GMIs) that ranged
from 6.4 to 12.2 (reference range, <0.5). Two dogs with local-
ized pulmonary disease had serum GMIs of 0.14 and 0.77, which
­suggests that, as for SNA, the sensitivity of this assay for diag-
nosis of bronchopulmonary aspergillosis may be low. High-level
644 SECTION 3  Fungal and Algal Diseases
A B
FIGURE 65-10  A, Morphology of Aspergillus fumigatus. B, Morphology of Aspergillus terreus, which has an “upswept” appearance. The conidiophore is the specialized hyphal branch
that bears the conidia (spores). The vesicle is the dilated top part of the conidiophore. Phialides are flask-shaped projections from the vesicle from which the conidia arise.
false positive test results (GMIs >1.5) appear to only occur in
dogs with other fungal infections, especially those caused by
Penicillium or Paecilomyces, and dogs treated with Plasmalyte
intravenous fluid therapy. Plasmalyte contains sodium gluconate,
which is produced by fermentation in ­Aspergillus niger; carry-
over of small amounts of galactomannan during production of
the fermentation product is thought to occur.52 Low-level false
positives (0.5-1.5) can occur in dogs infected with other fungi
(such as Cryptococcus spp.), some dogs with nonfungal disease
such as neoplasia, or some dogs treated with penicillin deriva-
tives. In summary, a strong positive assay result in a dog with
clinical abnormalities consistent with disseminated aspergillosis
and no history of Plasmalyte administration within the last 72
hours supports a diagnosis of a mold infection, but fungal culture
is required to confirm that Aspergillus is the causative agent. A
negative result in a dog with disseminated disease suggests that
Aspergillus is not the cause, but it does not rule out the possibility
of infection with another mold species.
Assays that detect antibodies to Aspergillus species are insen-
sitive for diagnosis of systemic aspergillosis.7 This may reflect
underlying host immunodeficiency (and hence inability to produce
antibodies), or antibodies in affected dogs may not cross-react
with the antigens used in the test kits. For example, one widely
used gel ID assay (Meridian Bioscience) contains a purified carbo-
hydrate preparation from mycelial phase cultures of A. fumigatus,
A. niger, and A. flavus, but not A. terreus or A. deflectus.
Molecular Diagnosis Using the Polymerase Chain Reaction
Several real-time PCR assays have been developed and applied
for the diagnosis of invasive aspergillosis in humans.53,54 The
sensitivity and specificity of these assays has varied.54 To date,
they have not been widely applied to diagnosis of disseminated
aspergillosis in dogs.
Pathologic Findings
Gross pathologic findings in dogs with disseminated aspergillo-
sis include enlarged and/or congested lymph nodes; multifocal,
pale, tan to white masses or nodules within a variety of paren-
chymal organs or the pleura (Figure 65-11, A); evidence of
discospondylitis and/or osteomyelitis; serosanguineous pleu-
ral effusion; and/or mottled and congested lung lobes. Splenic
infarction is also a common finding. The renal pelvis may be
dilated and contain soft, granular material. Histopathology
reveals granulomatous or pyogranulomatous inflammation
with abundant intralesional fungal hyphae in a variety of tis-
sues. Some species, such as A. terreus, also produce accessory
conidia (aleuriospores) in tissues, which may be free or extend
from hyphae. Fibrinoid necrosis of blood vessels and associated
hemorrhage may be present. Special stains such as Gomori’s
methenamine silver or periodic acid–Schiff can delineate fungal
hyphae in tissues (Figure 65-11, B). Immunohistochemistry has
also been used to identify Aspergillus.55,56
Treatment and Prognosis
Many dogs with disseminated aspergillosis have advanced disease
at the time of evaluation, sometimes with respiratory distress,
pathologic fractures, and vertebral subluxations and cord com-
pression. The prognosis for these dogs is poor. In one study, 57%
of 30 dogs were euthanized within a week of examination (median,
3 days).7 However, dogs that lack severe CNS signs, pain, or respi-
ratory distress may enter remission for months and occasionally
years when treated with antifungal drugs. These dogs maintain
high antigen titers or persistently positive urine cultures, and they
ultimately relapse and die from the disease. Sometimes, euthanasia
is performed earlier because continued treatment is unaffordable.
The relative efficacy of different antifungal drug treatment
regimens for disseminated aspergillosis in dogs has not been
studied prospectively. The most widely used treatments have
been itraconazole, voriconazole, posaconazole, and/or ampho-
tericin B. Terbinafine has been used in addition to azole therapy
with limited success in the author’s experience.7 Aspergillus spe-
cies are intrinsically resistant to fluconazole, so the use of fluco-
nazole is not recommended. Although expensive, treatment with

B
FIGURE 65-11  A, Kidney at necropsy from a 5-year-old female English setter with
disseminated aspergillosis caused by Aspergillus terreus. Numerous small coalescing tan
nodules are present throughout the cortex, and the capsular surface is irregular. There is
a blood clot adjacent to the renal pelvis. (Image courtesy University of California, Davis,
Veterinary Anatomic Pathology Service.) B, Histopathology from a 5-year-old male neu-
tered German shepherd dog with disseminated A. terreus infection that involved multiple
parenchymal organs. Large numbers of septate, branching hyphae are seen. Gomori’s
methenamine silver stain, 1000× oil magnification.
voriconazole or posaconazole can result in remissions of many
months’ duration in some dogs.7,57 Posaconazole in particular
has shown promising activity in human patient and mouse mod-
els of invasive A. terreus infection.58,59 Many dogs require treat-
ment with multiple antifungal drugs to maintain remission, either
in combination or in series. A. terreus strains from humans with
invasive aspergillosis can become resistant to azoles during treat-
ment.60 A. terreus strains isolated from human patients are also
relatively resistant to amphotericin B,59,61 but clinical improve-
ment can still occur after treatment of infected dogs with ampho-
tericin B (see Chapter 9).7 Echinocandins (such as caspofungin)
are also active against Aspergillus, but expense and the need for
intravenous administration has limited their use in dogs.
Dogs with localized bronchopulmonary aspergillosis have
the best prognosis. These dogs are sometimes cured after treat-
ment with itraconazole alone. Lung lobectomy may be required
CHAPTER 65  Aspergillosis 645
FIGURE 65-12  Severe ulcerative keratitis in a 12-year-old female spayed domestic
shorthair caused by an Aspergillus fumigatus complex organism. The condition was suc-
cessfully treated using topical voriconazole and a conjunctival island graft. (Courtesy the
University of California, Davis, Veterinary Ophthalmology Service.)
for successful treatment of German shepherds with cavitary
lung lesions.7,45,46
Immunity and Vaccination
Immunity to disseminated aspergillosis is critically dependent
on the function of neutrophils and dendritic cells, as well as
effective T cell function.62 A vaccine to prevent the disease is
not available.
Public Health Aspects
In human patients, invasive aspergillosis primarily occurs in
immunosuppressed humans with hematologic malignancy, or
after solid organ transplantation or hematopoietic stem cell
transplantation.51 Infections are acquired from the environment.
Transmission from affected dogs has not been described. Most
cases (>70%) are caused by A. fumigatus. Infection with A. ­terreus
is rare, and A. deflectus is not a recognized human pathogen.
KERATOMYCOSIS AND OTOMYCOSIS
CAUSED BY ASPERGILLUS SPECIES
Infection of the cornea8,63 and ear canal9,10 by Aspergillus spe-
cies occur rarely in dogs and cats (see Table 65-1; Figure 65-12).
Diagnosis of these conditions requires visualization of hyphae on
cytologic examination or histopathology of biopsy specimens in
addition to fungal culture to rule out the possibility of contami-
nation or colonization. One cat with A. flavus keratomycosis had
a history of feline herpesvirus-1–associated conjunctivitis and
keratitis8 and was successfully treated with 1% topical voricon-
azole (q2h to q4h). Cats with Aspergillus otomycosis often have
a history of diabetes mellitus.4 Dogs with otomycosis may have
a history of underlying allergic dermatitis and otitis externa that
has been extensively treated with topical and oral antibiotics.10
646 SECTION 3  Fungal and Algal Diseases
CASE EXAMPLE
Signalment: “Zach,” a 2-year-old male neutered German
shepherd from Roseville in northern California
History: Zach was evaluated at the University of California,
Davis, Veterinary Oncology Service for possible bone cancer.
Two months previously, the owners noted that Zach vocalized
when he stood up from rest. He was taken to a local veterinary
clinic where he was diagnosed with lumbosacral pain and
treated with carprofen (1.5 mg/kg PO q12h). Two weeks later,
right pelvic limb lameness developed. Pelvic radiographs
showed evidence of moderate osteoarthrosis of the left
coxofemoral joint. Carprofen treatment was continued. After
an additional 2 weeks, lethargy, inappetence, increased thirst
and urination, and left thoracic limb lameness developed,
and Zach began vomiting once daily. Radiographs of the
left thoracic limb revealed an osteoproductive lesion in the
mid-diaphysis of the left humerus, which was interpreted as
a primary or metastatic bone tumor (see Figure 65-8). Zach
was one of two dogs in the household, was normally fed a dry
commercial dog food diet, and was up to date on vaccinations,
which included those for distemper, adenovirus, parvovirus,
rabies, Borrelia, and Bordetella.
Physical Examination:
Body Weight: 33.5 kg.
General: Quiet, alert, and responsive. T = 103.4°F (39.7°C), HR
= 120 beats/min, panting, CRT <2 s, mucous membranes
moist and pink.
Eyes, Ears, Nose, and Throat: No clinically significant
abnormalities were detected. Fundoscopic examination
revealed multiple hyporeflective regions, consistent with
chorioretinitis, in the right fundus.
Musculoskeletal: BCS was 4/9, and generalized muscle
wasting was present. There was no evidence of lameness or
bone pain on orthopedic examination.
Cardiovascular, Respiratory, Gastrointestinal, Genitouri-
nary, and Lymph Nodes: No clinically significant abnor-
malities were present.
Laboratory Findings:
CBC:
HCT 32.8% (40%-55%)
MCV 65.2 fL (65-75 fL)
MCHC 35.7 g/dL (33-36 g/dL)
WBC 18,530 cells/µL (6000-13,000 cells/µL)
Neutrophils 13,768 cells/µL (3000-10,500 cells/µL)
Lymphocytes 2687 cells/µL (1000-4000 cells/µL)
Monocytes 1334 cells/µL (150-1200 cells/µL)
Eosinophils 741 cells/µL (0-1500 cells/µL)
Platelets 262,000 platelets/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 152 mmol/L (145-154 mmol/L)
Potassium 3.8 mmol/L (4.1-5.3 mmol/L)
Chloride 113 mmol/L (105-116 mmol/L)
Bicarbonate 24 mmol/L (16-26 mmol/L)
Phosphorus 5.7 mg/dL (3.0-6.2 mg/dL)
Calcium 11.8 mg/dL (9.9-11.4 mg/dL)
BUN 31 mg/dL (8-21 mg/dL)
Creatinine 1.9 mg/dL (0.5-1.6 mg/dL)
Glucose 107 mg/dL (60-104 mg/dL)
Total protein 6.8 g/dL (5.4-7.4 g/dL)
Albumin 2.8 g/dL (2.9-4.2 g/dL)
Globulin 4.0 g/dL (2.3-4.4 g/dL)
ALT 15 U/L (19-67 U/L)
AST 27 U/L (21-54 U/L)
ALP 28 U/L (15-127 U/L)
GGT 2 U/L (0-6 U/L)
Cholesterol 205 mg/dL (135-345 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.4 mg/dL).
Urinalysis: SGr 1.010; pH 7.0, 1+ protein (SSA), no bilirubin, 3+
hemoprotein, no glucose, 10-15 WBC/HPF, 50-60 RBC/HPF,
0-2 transitional epithelial cells, a few amorphous crystals,
and a few budding organisms were seen.
Imaging Findings:
Thoracic Radiographs: The cardiopulmonary structures were
unremarkable.
Bone Survey Radiographs: Single lateral radiographs of
the distal left antebrachium, distal right antebrachium,
and distal left and right pelvic limbs were performed. An
additional lateral radiograph of the left humerus was also
obtained and compared with referral radiographs taken 1
month previously. This showed progression of a primarily
osteoproductive lesion in the mid-diaphysis of the left
humerus. An additional site of chronic active periosteal
reaction was noted along the distal caudal aspect of the left
femur. Patchy increases in medullary density were identified
in the distal diaphysis of both humeri.
Abdominal Ultrasound: Hypoechoic mottling of the splenic
hilus was identified. There was bilateral renal pyelectasia
with echogenic material within the renal pelves bilaterally
(see Figure 65-9). There was marked blunting of the renal
pelves bilaterally with mild hydroureter.
Microbiologic Findings: Cytologic examination of a fine needle
aspirate of lesion in left humerus: Smears were moderately
cellular with a dense, proteinaceous background and
moderate amounts of cellular debris. Nucleated cells consisted
of a population of numerous activated macrophages and
variably degenerate neutrophils that were evenly distributed
throughout the smear as well as in variably sized aggregates.
Numerous multinucleated giant cells were noted. Throughout
the specimen were moderate numbers of small, round, yeast
organisms as well as numerous fungal hyphae, which were
found intracellularly and extracellularly. The fungal hyphae were
negative staining, branching, and septate, often with bulging
ends.
Fungal culture and susceptibility (left humerus fine needle aspi-
rate, sent to University of Texas for molecular identification):
Moderate numbers of Aspergillus terreus. MICs for ampho-
tericin B, itraconazole, and voriconazole were 2, 0.125, and
0.25 µg/mL, respectively.
Aerobic urine bacterial culture: Six colonies of A. terreus.
Aspergillus antibody serology (gel immunodiffusion, Meridian
Bioscience, Ltd.): Negative.
Diagnosis: Disseminated Aspergillus terreus infection.
Treatment: Zach was treated with intravenous fluids (lactated
Ringer’s solution with 20 mEq/L KCl at 75 mL/hr), lipid-
complexed amphotericin B (1 mg/kg IV on a Monday-
Wednesday-Friday basis for 4 weeks), and itraconazole
(5 mg/kg PO q12h). Over the course of amphotericin B
treatment, Zach’s inappetence, lethargy, and lameness
resolved and his serum creatinine and BUN concentrations
remained stable with values of 2.1 mg/dL and 49 mg/dL,

respectively, by the end of treatment. At a recheck 2 months
after treatment was initiated, fungal culture of the urine
revealed a single colony of A. terreus, and the creatinine was 1.7
mg/dL. After an additional month, bone survey radiographs
showed significant remodeling of the left humeral and left
femoral lesions. A serum biochemistry panel showed mildly
increased creatinine concentration (1.8 mg/dL) and a serum
ALT activity of 364 U/L; the increased serum ALT activity
was thought to be secondary to itraconazole administration.
Ultrasound of the abdomen showed mild pelvic dilatation,
and the echogenic material within the renal pelves had
disappeared. Fungal culture of the urine was negative. Four
months after treatment was initiated, intermittent vocalization
and right pelvic limb lameness developed. Pain was detected
only on lumbosacral palpation. Radiographs of this region
revealed focal osteolysis of the ventral caudal aspect of the
sixth lumbar vertebra. Clinical improvement did not occur
after treatment with carprofen and tramadol.
Two weeks later, Zach developed respiratory distress and was
seen by the Veterinary Emergency and Critical Care Service.
Mild dehydration, pyrexia (105.8°F or 41.0°C), gagging, and
ptyalism were detected on physical examination. Thoracic
radiographs were unremarkable, but a soft tissue density was
seen in the pharyngeal region on cervical radiographs. Blood
cultures were negative. Zach was sedated, and on oral exami-
nation, multiple large soft tissue masses were observed within
the pharynx and around the rim of the epiglottis. Aspiration
SUGGESTED READINGS
Barrs VR, Halliday C, Martin P, et al. Sinonasal and sino-orbital asper-
gillosis in 23 cats: aetiology, clinicopathological features and treat-
ment outcomes. Vet J. 2012;191:58-64.
Day MJ. Canine sino-nasal aspergillosis: parallels with human disease.
Med Mycol. 2009;47(suppl 1):S315-S323.
Schultz RM, Johnson EG, Wisner ER, et al. Clinicopathologic and diag-
nostic imaging characteristics of systemic aspergillosis in 30 dogs.
J Vet Intern Med. 2008;22:851-859.
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treatment outcomes. Vet J. 2012;191:58-64.
4. Sykes JE. Unpublished observations, 2012.
5. Kano R, Itamoto K, Okuda M, et al. Isolation of Aspergillus uda-
gawae from a fatal case of feline orbital aspergillosis. Mycoses.
2008;51:360-361.
6. Southard C. Bronchopulmonary aspergillosis in a dog. J Am Vet
Med Assoc. 1987;190:875-877.
7. Schultz RM, Johnson EG, Wisner ER, et al. Clinicopathologic and
diagnostic imaging characteristics of systemic aspergillosis in 30
dogs. J Vet Intern Med. 2008;22:851-859.
8. Labelle AL, Hamor RE, Barger AM, et al. Aspergillus flavus kera-
tomycosis in a cat treated with topical 1% voriconazole solution.
Vet Ophthalmol. 2009;12:48-52.
9. Ghibaudo G, Peano A. Chronic monolateral otomycosis in a dog
caused by Aspergillus ochraceus. Vet Dermatol. 2010;21:522-526.
CHAPTER 65  Aspergillosis 647
of the masses revealed suppurative inflammation and possible
occasional fungal hyphae. Bronchoscopy was performed, and
numerous yellow plaque-like lesions were visualized on the
tracheal mucosa. Cytologic examination of biopsies of these
lesions revealed fungal hyphae. The owner elected euthana-
sia. Necropsy findings included severe, locally extensive mural
granulomas of the tracheal mucosa with intralesional fungal
hyphae and suppurative and eosinophilic pharyngitis with
necrosis and edema. There was evidence of healed pyelone-
phritis and granulomatous nephritis with intralesional fungal
hyphae. Granulomatous osteomyelitis with intralesional hy-
phae was present in the left femur and humerus. There were
multiple granulomas in the pancreas and spleen.
Comments: Disseminated aspergillosis in this dog was initially
confused with bone neoplasia. An Aspergillus antibody titer
was negative, which illustrates the lack of sensitivity of this
assay for diagnosis of disseminated disease. Galactomannan
antigen testing was not performed, but bone lesions were
readily accessible for aspiration and culture. The small, round
yeast organisms visualized together with the hyphae were
likely accessory conidia, which can be found in A. terreus
infections. Treatment with lipid-complexed amphotericin
B and itraconazole was initially associated with clinical
remission, but infection was not eliminated. Based on
necropsy examination, the cause of the suppurative
pharyngeal masses that impaired respiration was not clear.
Fungal cultures of the masses were negative.
10. Coyner K. Otomycosis due to Aspergillus spp. in a dog: case report
and literature review. Vet Dermatol. 2010;21:613-618.
11. Zhang S, Corapi W, Quist E, et  al. Aspergillus versicolor, a new
causative agent of canine disseminated aspergillosis. J Clin Micro-
biol. 2012;50:187-191.
12. Barrs VR, van Doorn T, Houbraken J, et  al. Aspergillus felis sp.
nov. – an emerging pathogen of cats, dogs and humans. Proceed-
ings of the International Society for Companion Animal Infectious
Diseases. 2012: Abstract 017.
13. Talbot JJ, Martin P, Johnson LR, et  al. What causes sinonasal
aspergillosis in dogs? A molecular approach to species identifica-
tion. Proceedings of the International Society for Companion Ani-
mal Infectious Diseases. 2012: Abstract 018.
14. Burrough E, Deitz K, Kinyon J, et al. Disseminated aspergillosis in
a dog due to Aspergillus alabamensis. Med Mycol Case Reports.
2012;1:1-4.
15. Pitt JI, Samson RA. Nomenclatural considerations in naming spe-
cies of Aspergillus and its teleomorphs. Stud Mycol. 2007;59:67-70.
16. Pomrantz JS, Johnson LR, Nelson RW, et al. Comparison of sero-
logic evaluation via agar gel immunodiffusion and fungal culture
of tissue for diagnosis of nasal aspergillosis in dogs. J Am Vet Med
Assoc. 2007;230:1319-1323.
17. Day MJ. Canine sino-nasal aspergillosis: parallels with human dis-
ease. Med Mycol. 2009;47(suppl 1):S315-S323.
18. Goodall SA, Lane JG, Warnock DW. The diagnosis and treat-
ment of a case of nasal aspergillosis in a cat. J Small Anim Pract.
1984;25:627-633.
19. Peeters D, Day MJ, Clercx C. An immunohistochemical study of
canine nasal aspergillosis. J Comp Pathol. 2005;132:283-288.
20. Peeters D, Peters IR, Clercx C, et  al. Quantification of mRNA
encoding cytokines and chemokines in nasal biopsies from dogs
with sino-nasal aspergillosis. Vet Microbiol. 2006;114:318-326.
21. Hamilton HL, Whitley RD, McLaughlin SA. Exophthalmos second-
ary to aspergillosis in a cat. J Am Anim Hosp Assoc. 2000;36:343-347.
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22. Giordano C, Gianella P, Bo S, et al. Invasive mould infections of the
naso-orbital region of cats: a case involving Aspergillus fumigatus
and an aetiological review. J Feline Med Surg. 2010;12:714-723.
23. Barachetti L, Mortellaro CM, Di Giancamillo M, et  al. Bilat-
eral orbital and nasal aspergillosis in a cat. Vet Ophthalmol.
2009;12:176-182.
24. Billen F, Guieu LV, Bernaerts F, et  al. Efficacy of intrasinusal
administration of bifonazole cream alone or in combination with
enilconazole irrigation in canine sino-nasal aspergillosis: 17 cases.
Can Vet J. 2010;51:164-168.
25. Pomrantz JS, Johnson LR. Repeated rhinoscopic and serologic
assessment of the effectiveness of intranasally administered clotrim-
azole for the treatment of nasal aspergillosis in dogs. J Am Vet Med
Assoc. 2010;236:757-762.
26. Saunders JH, Clercx C, Snaps FR, et  al. Radiographic, magnetic
resonance imaging, computed tomographic, and rhinoscopic
features of nasal aspergillosis in dogs. J Am Vet Med Assoc.
2004;225:1703-1712.
27. Johnson LR, Drazenovich TL, Herrera MA, et  al. Results of rhi-
noscopy alone or in conjunction with sinuscopy in dogs with
aspergillosis: 46 cases (2001-2004). J Am Vet Med Assoc.
2006;228:738-742.
28. Whitney BL, Broussard J, Stefanacci JD. Four cats with fungal rhi-
nitis. J Feline Med Surg. 2005;7:53-58.
29. Peeters D, Clercx C. Update on canine sinonasal aspergillosis. Vet
Clin North Am Small Anim Pract. 2007;37:901-916, vi.
30. Smith LN, Hoffman SB. A case series of unilateral orbital aspergil-
losis in three cats and treatment with voriconazole. Vet Ophthal-
mol. 2010;13:190-203.
31. De Lorenzi D, Bonfanti U, Masserdotti C, et  al. Diagnosis of
canine nasal aspergillosis by cytological examination: a compari-
son of four different collection techniques. J Small Anim Pract.
2006;47:316-319.
32. Billen F, Clercx C, Le Garerres A, et al. Effect of sampling method
and incubation temperature on fungal culture in canine sinonasal
aspergillosis. J Small Anim Pract. 2009;50:67-72.
33. Billen F, Peeters D, Peters IR, et  al. Comparison of the value of
measurement of serum galactomannan and Aspergillus-specific
antibodies in the diagnosis of canine sino-nasal aspergillosis. Vet
Microbiol. 2009;133:358-365.
34. Peeters D, Peters IR, Helps CR, et al. Whole blood and tissue fungal
DNA quantification in the diagnosis of canine sino-nasal aspergil-
losis. Vet Microbiol. 2008;128:194-203.
35. Tomsa K, Glaus TM, Zimmer C, et al. Fungal rhinitis and sinusitis
in three cats. J Am Vet Med Assoc. 2003;222:1380-1384, 1365.
36. Garcia RS, Wheat LJ, Cook AK, et al. Sensitivity and specificity of
a blood and urine galactomannan antigen assay for diagnosis of
systemic aspergillosis in dogs. J Vet Intern Med. 2012;26:911-919.
37. Whitney J, Beatty JA, Martin P, et al. Evaluation of serum galac-
tomannan detection for diagnosis of feline upper respiratory tract
aspergillosis. Vet Microbiol. 2013;162:180-185.
38. Furrow E, Groman RP. Intranasal infusion of clotrimazole for the
treatment of nasal aspergillosis in two cats. J Am Vet Med Assoc.
2009;235:1188-1193.
39. McCullough SM, McKiernan BC, Grodsky BS. Endoscopically
placed tubes for administration of enilconazole for treatment of
nasal aspergillosis in dogs. J Am Vet Med Assoc. 1998;212:67-69.
40. Caulkett N, Lew L, Fries C. Upper-airway obstruction and pro-
longed recovery from anesthesia following intranasal clotrimazole
administration. J Am Anim Hosp Assoc. 1997;33:264-267.
41. Sissener TR, Bacon NJ, Friend E, et  al. Combined clotrimazole
irrigation and depot therapy for canine nasal aspergillosis. J Small
Anim Pract. 2006;47:312-315.
42. Hazell KL, Swift IM, Sullivan N. Successful treatment of pulmo-
nary aspergillosis in a cat. Aust Vet J. 2011;89:101-104.
43. Sykes JE, Mapes S, Lindsay LL, et  al. Corynebacterium ulcerans
bronchopneumonia in a dog. J Vet Intern Med. 2010;24:973-976.
44. Adamama-Moraitou KK, Pardali D, Day MJ, et  al. Aspergillus
fumigatus bronchopneumonia in a Hellenic shepherd dog. J Am
Anim Hosp Assoc. 2011;47:e13-e18.
45. Kulendra E, Halfacree Z, Goggs R, et  al. Cavitary pulmonary
lesion associated with Aspergillus fumigatus infection in a German
shepherd dog. J Small Anim Pract. 2010;51:271-274.
46. Whitley NT, Cauvin A, Burton C, et al. Long term survival in two
German shepherd dogs with Aspergillus-associated cavitary pulmo-
nary lesions. J Small Anim Pract. 2010;51:561.
47. Guerin SR, Walker MC, Kelly DF. Cavitating mycotic pneumonia
in a German shepherd dog. J Small Anim Pract. 1993;34:36-39.
48. Krockenberger MB, Swinney G, Martin P, et al. Sequential oppor-
tunistic infections in two German Shepherd dogs. Aust Vet J.
2011;89:9-14.
49. Fox JG, Murphy JC, Shalev M. Systemic fungal infections in cats.
J Am Vet Med Assoc. 1978;173:1191-1195.
50. Ossent P. Systemic aspergillosis and mucormycosis in 23 cats. Vet
Rec. 1987;120:330-333.
51. Steinbach WJ, Marr KA, Anaissie EJ, et al. Clinical epidemiology
of 960 patients with invasive aspergillosis from the PATH alliance
registry. J Infect. 2012;65:453-464.
52. Hage CA, Reynolds JM, Durkin M, et al. Plasmalyte as a cause of
false-positive results for Aspergillus galactomannan in bronchoal-
veolar lavage fluid. J Clin Microbiol. 2007;45:676-677.
53. White PL, Linton CJ, Perry MD, et  al. The evolution and evalu-
ation of a whole blood polymerase chain reaction assay for the
detection of invasive aspergillosis in hematology patients in a rou-
tine clinical setting. Clin Infect Dis. 2006;42:479-486.
54. Johnson GL, Bibby DF, Wong S, et al. A MIQE-compliant real-time
PCR assay for Aspergillus detection. PLoS One. 2012;7:e40022.
55. Butterworth SJ, Barr FJ, Pearson GR, et al. Multiple discospondy-
litis associated with Aspergillus species infection in a dog. Vet Rec.
1995;136:38-41.
56. Perez J, Mozos E, de Lara FC, et  al. Disseminated aspergillo-
sis in a dog: an immunohistochemical study. J Comp Pathol.
1996;115:191-196.
57. Legendre AM. Unpublished observations, 2012.
58. Salas V, Pastor FJ, Rodriguez MM, et  al. In  vitro activity and
in vivo efficacy of posaconazole in treatment of murine infections
by different isolates of the Aspergillus terreus complex. Antimicrob
Agents Chemother. 2011;55:676-679.
59. Hachem RY, Kontoyiannis DP, Boktour MR, et  al. Aspergil-
lus terreus: an emerging amphotericin B–resistant opportunis-
tic mold in patients with hematologic malignancies. Cancer.
2004;101:1594-1600.
60. Arendrup MC, Jensen RH, Grif K, et  al. In  vivo emergence of
Aspergillus terreus with reduced azole susceptibility and a Cyp51a
M217I alteration. J Infect Dis. 2012;206:981-985.
61. Escribano P, Pelaez T, Recio S, et  al. Characterization of clinical
strains of Aspergillus terreus complex: molecular identification and
antifungal susceptibility to azoles and amphotericin B. Clin Micro-
biol Infect. 2012;18:E24-E26.
62. Park SJ, Burdick MD, Mehrad B. Neutrophils mediate maturation
and efflux of lung dendritic cells in response to Aspergillus fumiga-
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63. Qualls Jr CW, Chandler FW, Kaplan W, et al. Mycotic keratitis in a
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Vet Med Assoc. 1985;186:975-976.
CHAPTER 66

Rhinosporidiosis
Jane E. Sykes

majority of affected dogs are young-adult to middle-aged (with

Overview of Rhinosporidiosis
First Described: In the 1890s, in Argentina (by Malbran and
then by Seeber)1
Cause: Rhinosporidium seeberi (kingdom Protista, class
Mesomycetozoea)
Affected Hosts: Dogs, humans, horses, rarely cats and other
domestic animals
Geographic Distribution: Worldwide but especially in warm,
wet environments such as the south-eastern and south-
central United States
Mode of Transmission: Unclear; possibly exposure to contam-
inated water sources in association with trauma
Major Clinical Signs: Sneezing, epistaxis, mass protruding
from nares
Differential Diagnoses: Nasal neoplasia, cryptococcosis, sino-
nasal aspergillosis, nasal mites (Pneumonyssoides cani-
num), foreign bodies
Human Health Significance: Human infections by R. seeberi
are likely acquired from the environment, and direct
transmission from affected animals to humans has not
been described.
Etiology and Epidemiology
Rhinosporidiosis is caused by Rhinosporidium seeberi, which
causes slow-growing tumor-like masses in the nasal cavity.
Although once thought to be a fungus, molecular methods
have identified this organism as an aquatic protistan parasite
(class Mesomycetozoea). The organism is taxonomically located
where animals and fungi diverge and is closely related to patho-
gens of fish.2 The organism is difficult or impossible to culture.
Rhinosporidiosis has been most widely reported in humans and
dogs, but cats, horses, and other mammalian and avian species
may also be affected. There is some evidence that host-specific
strains of R. seeberi exist.3
In humans, rhinosporidiosis is most prevalent in southern
India, Sri Lanka, and Argentina but also occurs in North Amer-
ica, Africa, Europe, and Asia. In dogs and cats, rhinosporidi-
osis has been reported from many regions of the United States,
Ontario in Canada,4 Argentina,5 Italy in continental Europe,6
and the United Kingdom.7 Within the United States, the disease
is most often reported in dogs and cats from the south-
­central and southeastern states (Figure 66-1). The overwhelming
a range of 1 to 13 years of age), otherwise-healthy, large-breed
hunting dogs such as Labrador and golden retrievers, Doberman
pinschers, Siberian huskies, German shepherds, and Rhodesian
ridgebacks. However, small-breed dogs can also be affected.7
No clear sex predisposition has been identified, but of cases
in the literature, males have outnumbered females. Affected
cats have been outdoor cats. One affected cat had concurrent
nasal adenocarcinoma,8 but other cats have had no reported
comorbidities.
Although the mode of transmission of R. seeberi is not pre-
cisely understood, disease often occurs in association with con-
tact with aquatic or marshy environments. It is thought that
spores are released into the environment, where they contact
susceptible human and animal hosts.2 Spores may be introduced
into tissues after trauma to the mucous membranes, where they
form sporangia that produce new spores. Because disease in
humans also occurs in arid regions, airborne spore transmission
has also been suggested.
Clinical Features
Signs and Their Pathogenesis
In dogs and cats, infection by R. seeberi leads to formation of
pedunculate or sessile masses within the nasal cavity, usually
in the rostral third.6 The polyps range in size from a few mil-
limeters to several centimeters. Clinical signs consist of sub-
acute (weeks) to chronic (>1 year) sneezing with or without
epistaxis, snuffling, and sometimes a serous, mucoid, or sero-
sanguineous unilateral nasal discharge. Sneezing may be fre-
quent and severe and associated with excitement or increased
FIGURE 66-1  States within the United States in which rhinosporidiosis has been
reported in dogs and cats.

649
650 SECTION 3  Fungal and Algal Diseases

FIGURE 66-2  CT scan of the nasal cavity of a 16-month-old female Labrador retriever FIGURE 66-3  Rhinoscopic image of a polypoid mass caused by Rhinosporidium see-
with rhinosporidiosis. A soft tissue mass that enhanced with contrast is present in the beri in the nasal cavity of a 10-year-old intact female yellow Labrador retriever.
­rostral nasal cavity (arrow).

activity. In some dogs, a polypoid pink mass protrudes from

the nasal cavity. The masses are often stippled with white-
yellowish granules less than 1 mm in diameter, which represent
mature sporangia.6

Diagnosis
Most animals with rhinosporidiosis have no significant abnor-
malities on blood work. Plain radiography is insensitive for
detection of nasal masses caused by R. seeberi.6 Computed
tomography (CT) reveals soft tissue masses in the rostral third
of the nasal cavity that can enhance with contrast (Figure 66-2).
In some cases evidence of turbinate loss is present. The masses,
which can be visualized directly using rostral rhinoscopy, are
pink to gray and may be covered with miliary white to yellow
foci, but these are not always clearly visible (Figure 66-3).
Definitive diagnosis requires cytology or histopathology
of the masses. Cytologic examination of smears from nasal
swabs, cytology brush specimens, or impression smears made
from biopsies of the polyps reveals suppurative to pyogranu-
lomatous inflammation with dysplastic epithelial cells and
numerous immature to mature R. seeberi endospores, which
occur singly or in clusters.9 Sometimes sporangia that contain
hundreds of endospores are visualized. Mature endospores
stain intensely, are round to oval and 10 to 15 µm in diam-
eter, and have a thick cell wall (Figure 66-4). Immature endo-
spores are 2 to 4 µm in diameter and contain a paracentral,
light pink-purple structure that may represent nuclear mate-
rial, as well as one to two smaller, spherical, dark purple
structures.9
Histopathology reveals abundant sporangia in various stages
of development and a mild to severe pyogranulomatous inflam-
matory response (Figure 66-5). Lymphocytes, plasma cells, and,
in some cases, multinucleate giant cells and evidence of turbi-
nate remodeling with activated osteoclasts and osteoblasts may
be present. Juvenile sporangia are small (<100 µm in diameter)
and have a single nucleus that is surrounded by fibrillar mate-
rial. Mature sporangia are up to 500 µm in diameter and may
FIGURE 66-4  Impression smear of a biopsy taken from the nasal cavity of a 6-year-
old female spayed Labrador retriever with rhinosporidiosis. A large cluster of endospores is
present together with several epithelial cells.
be seen releasing endospores at the polyp surface through a pore
in the sporangial wall. Although the organisms stain well with
special stains such as periodic acid–Schiff, Gomori’s methena-
mine silver, and Mayer’s mucicarmine, these stains are usually
not required for diagnosis.6
Treatment and Prognosis
The treatment of choice for dogs and cats is surgical removal
of polyps, which generally requires rhinotomy. Because the
polyps are often rostrally located, excisions can often be
extended caudally from the nares. Many dogs have no recur-
rence of clinical signs thereafter, but some dogs develop
relapse within months of surgery.5,10,11 Treatment of these
dogs can be frustrating and costly. Povidone-iodine is active
against R. seeberi in  vitro and could be applied in the form
of gauze packs topically after surgery in an attempt to mini-
mize the chance of recurrence (see Case Example).12 In some
dogs, apparent transient or long-term responses to treatment
with ketoconazole7,13 or dapsone (1.1 mg/kg PO q8-12h)11

A B
FIGURE 66-5  Histopathology of a polyp caused by Rhinosporidium seeberi. A, Low-power image that shows sporangia in various stages of development. B, High-power image
(1000× oil magnification) that shows a juvenile sporangium adjacent to a mature sporangium, which contains endospores (right). H&E stain.
have been described. Responses to dapsone have also been
described in human patients. Dapsone could be used to treat
dogs with refractory disease, but because both dapsone and
ketoconazole can have significant adverse effects, careful mon-
itoring is required. Dapsone is a sulfur drug (diaminophenyl
sulfone) that can cause bone marrow suppression in dogs14
and has the potential to cause hepatotoxicity and cutaneous
drug reactions. Treatment of cats with dapsone is not recom-
mended because of a high prevalence of adverse effects in this
species, specifically neurotoxicosis and anemia.
CASE EXAMPLE
Signalment: “Sarah,” a 16-month-old intact female Labrador
retriever from the central valley of northern California
History: Sarah was evaluated by her local veterinary clinic for
a 1-month history of sneezing, which occurred throughout
the day and was more frequent and severe during periods
of excitement. The owners had not observed nasal
discharge, and they were concerned about the possibility of
a foreign body. A limited rhinoscopic examination with an
otoscope cone at the local veterinary clinic revealed a large,
fleshy, irregular mass in the dorsal meatus near the nares.
Histopathology on a biopsy of the mass yielded a diagnosis
of rhinosporidiosis, and Sarah was referred to the University
of California, Davis, Veterinary Medical Teaching Hospital for
further evaluation and treatment.
Sarah had been obtained from a breeder in Oregon as a puppy
and since then had been a duck hunting dog in California.
During the hunting season, she waded and swam in stag-
nant and fresh water sources. The owners also had ponds in
their backyard. She was otherwise healthy and was fed a dry
commercial dog food diet.
CHAPTER 66  Rhinosporidiosis 651
Public Health Aspects
In humans, rhinosporidiosis usually involves the nasal cavity
or nasopharynx, but involvement of the conjunctiva, genita-
lia, or skin can occur.15 Rarely, cutaneous disease is dissemi-
nated. Disease is most often reported in males. Infection is likely
acquired from the environment, and transmission from animals
to humans has not been described. It is possible that strains of
R. seeberi that infect humans may differ from those that infect
dogs.
Physical Examination:
Body Weight: 32.2 kg.
General: Excited, alert, responsive, and hydrated. T = 101.6°F
(38.7°C), HR = 140 beats/min, panting, CRT <2 s, mucous
membranes moist and pink.
Eyes, Ears, Nose, and Throat: The dog’s face was symmetrical
with no pain on palpation, and normal ocular retropulsion.
There was no evidence of nasal discharge. Nasal airflow
was absent on the right side. Minimal dental calculus was
present. There were no other clinically significant findings.
Musculoskeletal: BCS 4/9. Adequate musculature, normal gait.
Cardiovascular, Respiratory, Gastrointestinal, Genitourinary,
and Lymph Nodes: No clinically significant abnormalities
were detected.
Laboratory Findings: Preanesthetic laboratory assessment:
PCV 44%, total solids 6.6 g/dL. Serum urea nitrogen was
estimated at 15-26 mg/dL with a reagent strip.
Imaging Findings: Computed tomography of the head: A
round, 0.9 × 1.7 × 2.3 cm, soft tissue mass was present in the
rostral, dorsal right nasal cavity extending from the level of
the incisors to the root of the right maxillary canine tooth.
The mass was moderately and heterogeneously contrast
enhancing. The superficial soft tissues overlying the mass
Continued
652 SECTION 3  Fungal and Algal Diseases
were thickened and contrast enhancing. The remainder of
the nasal cavities and sinuses appeared within normal limits.
The mandibular and medial retropharyngeal lymph nodes
appeared within normal limits.
Diagnosis: Nasal rhinosporidiosis.
Treatment: A rhinotomy was performed using an anterolateral
approach, and a single 1.5 cm × 0.5 cm pedunculated mass
was identified attached to the mucosal layer of the nasal
septum. The mass was resected with sharp dissection and
diathermy. A povidone-iodine–soaked gauze pad was
placed on the site of resection for 7 minutes, and the nasal
vestibule was packed with an iodine-soaked gauze pad for
1 hour after surgical closure. Histopathology of the resected
mass confirmed the diagnosis of nasal rhinosporidiosis.
SUGGESTED READING
Caniatti M, Roccabianca P, Scanziani E, et al. Nasal rhinosporidiosis in
dogs: four cases from Europe and a review of the literature. Vet Rec.
1998;142:334-338.
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the dog. Vet Pathol. 1986;23:50-56.
14. Lees GE, McKeever PJ, Ruth GR. Fatal thrombocytopenic hemor-
rhagic diathesis associated with dapsone administration to a dog.
J Am Vet Med Assoc. 1979;175:49-52.
15. Prasad K, Veena S, Permi HS, et al. Disseminated cutaneous rhino-
sporidiosis. J Lab Physicians. 2010;2:44-46.
CHAPTER 67
Candidiasis
Jane E. Sykes
Overview of Candidiasis
First Described: The etiology of candidiasis as a fungus was
recognized in Stockholm around 1840 (Fredrik Berg),1 but
the disease was described as far back as around 400 bc by
Hippocrates.
Cause: Candida spp. (an ascomycete)
Affected Hosts: Dogs, cats, humans, and a variety of other
animal species
Geographic Distribution: Worldwide
Mode of Transmission: Usually invasion of commensal yeasts
secondary to immune suppression
Major Clinical Signs: Local dermatitis; otitis externa; keratitis;
lower urinary tract signs; abdominal pain, icterus, and/or
ascites due to peritonitis; or severe systemic infections
with fever, inappetence, ocular inflammation, lameness
due to osteomyelitis, respiratory distress, vomiting
Differential Diagnoses: Other systemic fungal or bacterial
infections
Human Health Significance: Human infections by Candida
species result from invasion by normal flora, but human-
to-human transmission of Candida strains can also occur.
Whether resistant Candida species can be transferred from
animals to colonize humans or vice versa is not known.
Etiology and Epidemiology
Candida species are yeasts that are typically benign counterparts
of the normal gastrointestinal, urogenital, and cutaneous flora
but can invade tissues and cause disease as a result of disrup-
tion of normal host defenses. Candida species can also be iso-
lated from soil, inanimate objects, and hospital environments.
The yeasts are small (3 to 6 µm) and ovoid (blastospores) and
reproduce by budding. Budding results in the formation of new
yeast cells, pseudohyphae (chains of elongated yeast cells), and
true septate hyphae. Disease syndromes caused by Candida spe-
cies that occur in dogs and cats are listed in Box 67-1. More than
200 species of Candida exist, but only a few species have been
identified as pathogens in dogs and cats (Table 67-12-10). The
most commonly isolated species is Candida albicans, and only
C. albicans appears to cause disseminated disease in dogs and
cats. Local or disseminated infections where Candida is present
with a mixture of bacterial species can occur. Local infections
(such as UTIs) with two different Candida species have also been
reported.2,11
BOX 67-1
Disease Syndromes Caused by Candida in Dogs and Cats
Keratitis
Cutaneous candidiasis
Otitis externa and otitis media
Urinary tract infections
Gastrointestinal candidiasis
Peritonitis
Disseminated infections
When compared with human patients, candidiasis is
uncommonly described in dogs and cats. Predisposing factors
in dogs or cats with local or disseminated candidiasis have
included diabetes mellitus,2,3,12 treatment with immunosup-
pressive drugs or broad-spectrum antibacterial drugs,2,13 a his-
tory of gastrointestinal surgery,4,5 parvoviral infections,14-16
and/or underlying malignancy.2,17 Often more than one of
these factors is in the history. Disseminated candidiasis has
most often been described in dogs, but also occurs in cats.5,18
Although most animals have underlying immunocompromis-
ing conditions, disseminated candidiasis has been described
in apparently immunocompetent animals,19,20 which may
reflect an unidentified underlying genetic immunodeficiency.
Cats with candidiasis usually test negative for FeLV or FIV
infection.
Analysis of the author’s hospital population suggests that
cats are at least as likely as dogs to develop Candida UTIs.11
In other studies, Candida species were isolated from the urine
of 0.2% of more than 8000 dogs with UTIs21 and 0.2% of
more than 2000 dogs with persistent or recurrent UTIs.22 Ure-
throstomy or indwelling cystostomy tube placement may be in
the history of some cats and dogs with Candida UTIs, respec-
tively.2,6 Local mucocutaneous infections can occur at the site of
long-term placement of feeding tubes.7
Clinical Features
Signs and Their Pathogenesis
Disruption of normal barriers is necessary to allow Candida spp.
to invade the host. Concurrent use of broad-spectrum antibiot-
ics suppresses the normal bacterial flora and allows Candida to
proliferate, which increases the risk that organisms will be intro-
duced into tissues. Candida can adhere to a variety of tissue com-
ponents such as epithelial and endothelial cells, fibronectin, and
653
654 SECTION 3  Fungal and Algal Diseases

thrombi, as well as other bacteria and inanimate objects such as in the immune response to Candida.24 The formation of hyphae
catheters.23 The yeasts can also form biofilms on medical devices by Candida promotes tissue invasion, which may be followed by
and produce a huge variety of hydrolytic enzymes. Within tis- life-threatening fungemia and dissemination to multiple organs.
sues, neutrophils are a key defense against Candida.24 Candida This ability of Candida to change its morphology (“morphologic
blastospores are phagocytized and destroyed by neutrophils. dimorphism”) is thought to be an important virulence factor.
Mononuclear cells play a less important role. Reactive oxygen Lower urinary tract candidiasis may be subclinical or asso-
species, hydrolytic enzymes, and antimicrobial peptides contrib- ciated with dysuria, stranguria, and/or hematuria. Systemic
ute to the intracellular destruction of blastospores by neutro- signs of pyrexia, lethargy, inappetence, and weight loss have
phils. Thus, any condition that impairs neutrophil numbers or also been described in affected dogs and cats,6 but these signs
function, such as neutropenia or diabetes mellitus, predisposes may result from the presence of other predisposing diseases.
dogs and cats to candidiasis. A large number of other cytokines, Ascending infections may lead to development of Candida
complement, dendritic cells, and lymphocytes are also involved pyelonephritis.
Cutaneous candidiasis may manifest as erythema, scaling,
alopecia, erosions, and crusting, often where disruption of
cutaneous or mucocutaneous regions has occurred (such as a
TABLE 67-1 previous surgical site or a feeding tube or cystotomy stoma).
Candida Species Associated with Disease in Dogs and/or Cats 2-10 Persistent disease in the face of systemic or topical treatment
with broad-spectrum antibiotics should raise suspicion for can-
Species Clinical Manifestations didiasis. Dogs with Candida otitis externa often have a history
of chronic bacterial otitis externa that has been treated aggres-
Candida albicans Dermatitis, UTI, otitis externa, sively with systemic or topical antibacterials.
keratitis, disseminated infections Overgrowth of Candida in the gastrointestinal tract may occur
Candida glabrata (formerly UTI, feeding-tube site infections in animals that are immunosuppressed or treated with glucocor-
Torulopsis glabrata) ticoids and/or broad-spectrum antibacterial drugs. In some cases,
Candida krusei UTI, feeding tube site infections invasion of the mucosa into the lamina propria or deeper tissues
occurs (e.g., intestinal candidiasis). Intestinal candidiasis is occa-
Candida guilliermondii UTI, dermatitis
sionally identified at necropsy in puppies that die after treatment
Candida parapsilosis UTI, otitis, dermatitis for parvoviral enteritis and may be a reason for persistent vomit-
Candida tropicalis UTI ing, diarrhea, and death despite aggressive fluid and antibacterial
Candida rugosa UTI drug therapy.14 Candida spp. may also contribute to ulcerative
glossitis in puppies with parvovirus infection (Figure 67-1).

A B
FIGURE 67-1  A, Tongue ulcerations at necropsy in a 2-month-old mixed-breed puppy that was euthanized because of canine parvoviral infection. B, Histopathology revealed mul-
tifocal ulcerative glossitis, pharyngitis, and esophagitis with intralesional yeasts and pseudohyphae. Gomori’s methenamine silver stain. (Image courtesy University of California Davis
Anatomic Pathology Service.)

Candida peritonitis can follow intestinal surgery, treatment
with broad-spectrum antibacterial drugs, and subsequent enter-
otomy site dehiscence.4,5 Mixed infections with Candida and
intestinal flora may develop. Affected dogs are febrile, have
abdominal pain, and may have other gastrointestinal signs such
as vomiting and diarrhea.
Disseminated candidiasis results from hematogenous
spread of Candida from intestinal, urinary, or possibly corneal
or cutaneous sites.12,15,25A diagnosis of disseminated candi-
diasis should be considered whenever Candida infections are
identified in the eye, skin, or urinary tract of an animal that
has (or subsequently develops) severe systemic illness. Clini-
cal signs of disseminated candidiasis in dogs include fever,
inappetence, anorexia, weight loss, and a variety of other
clinical signs that reflect underlying immunosuppressive dis-
ease processes and specific organs involved. The latter can
include the pancreas, liver, mesentery, spleen, kidneys, heart,
lungs, lymph nodes, and, less often, the bone, intestinal tract,
eyes, meninges, thyroid, and/or prostate gland.3,12,15,17,20,25-
28 Ocular signs in animals with disseminated disease include
keratitis and corneal ulceration, uveitis, chorioretinitis, and
endophthalmitis.11,12,28 Candida keratitis may also occur
as a localized process, sometimes secondary to aggressive
treatment of corneal ulceration secondary to other disease
processes with topical broad-spectrum antibacterial drugs.
Thromboembolic disease may complicate disseminated infec-
tions. Pulmonary thromboembolism may result in respiratory
distress and death.3,11
Diagnosis
Laboratory Abnormalities
Laboratory abnormalities in dogs and cats with candidiasis
result from the underlying disease process (such as diabetes
mellitus or systemic neoplastic disease) as well as the location
and severity of the fungal infection. Laboratory abnormalities
in dogs or cats with Candida UTIs may be absent, or there may
be evidence of renal failure with azotemia and isosthenuria.
A B
FIGURE 67-2  Candida albicans within urine sediment from a 5-year-old female spayed Labrador retriever being treated for multicentric lymphoma and acute-onset hematuria. Both
budding (A) and filamentous (B) fungal elements are visible. (Image courtesy Dr. Barrak Pressler, The Ohio State University. From Bartges J, Polzin D. Nephrology and Urology of Small
Animals. John Wiley and Sons, 2011.)
CHAPTER 67  Candidiasis 655
Grossly, urine may contain flocculent white debris. Urinalysis
may reveal proteinuria, pyuria, and, in some cases, hematuria
and/or Candida fungal elements (see Case Example). Dogs and
cats with disseminated candidiasis or Candida peritonitis fre-
quently have had nonregenerative anemia and mild to marked
neutrophilia with bandemia and toxic neutrophil changes (in
contrast to other deep mycoses, where hematologic abnormali-
ties are often mild or absent). Lymphopenia may be present as a
result of underlying immunosuppressive illness or drug therapy.
Some dogs are thrombocytopenic and have coagulation abnor-
malities consistent with disseminated intravascular coagula-
tion.3 Findings on the serum biochemistry panel in dogs with
disseminated candidiasis are variable and nonspecific but may
include metabolic acidosis, moderate to severe hypoalbumin-
emia, and increased liver enzyme activities.
Diagnostic Imaging
Plain thoracic radiographs in dogs with disseminated candidia-
sis may be unremarkable or show increased pulmonary inter-
stitial opacity, thoracic lymphadenomegaly, and/or pleural or
pericardial effusion.13,5,20 Abdominal ultrasound examination
in dogs with Candida peritonitis or disseminated disease may
reveal echogenic ascites fluid, mesenteric hyperechogenicity,
changes consistent with pancreatitis, abdominal lymphadeno-
megaly, or increased echogenicity of the renal cortices or hepatic
parenchyma.
Microbiologic Tests
Definitive diagnosis of candidiasis requires visualization of the
organism within lesions by cytologic or histopathologic exami-
nation and confirmation of its identity using culture.
Cytologic Examination
Candida blastospores, pseudohyphae, and true hyphae can be
visualized in swab specimens from the ear canal (ear ­cytology);
tape preparations from the skin; urine sediment or other
body fluids (Figure 67-2); or ultrasound-guided aspirates of
­abdominal lesions.
656 SECTION 3  Fungal and Algal Diseases
Fungal Culture
Candida grows on routine bacteriologic media as well as fungal
media such as Sabouraud dextrose agar. Creamy white colo-
nies appear within 2 to 3 days, and so isolation of the yeast
may be reported when it is present in specimens submitted for
aerobic bacterial culture, which are typically incubated only for
this period of time. If Candida is suspected based on the his-
tory, fungal culture (rather than bacterial culture alone) should
be requested because Candida grows best on fungal isolation
media. Because Candida can be isolated from the skin and
mucous membranes of healthy dogs and cats, infection must
be confirmed with cytology or histopathology when Candida is
isolated from these sites.
Identification of Candida isolates is based on morphology
and biochemical reactions. Preliminary differentiation of C.
albicans from other Candida species can be made based on
the ability of C. albicans to produce germ tubes when exposed
to serum. Matrix-assisted laser desorption/ionization–time of
flight (MALDI-TOF) mass spectrometry (see Chapter 3) accu-
rately and rapidly identifies Candida species29 and may replace
these methods in the future. Isolation of Candida allows not
only definitive identification but also antifungal susceptibility
testing (see Figure 4-8). This is important for Candida because
some isolates (especially C. glabrata and C. krusei) can exhibit
antifungal drug resistance.
Pathologic Findings
Gross pathologic findings at necropsy in animals with dissemi-
nated candidiasis include peritoneal or pleural effusion, enlarged
and edematous lymph nodes, congestion and necrotic whitish
foci within affected organs, evidence of tissue thrombosis, and
formation of a gray-white or yellow-green fibrinonecrotic layer
on affected mucosal surfaces.3,5,14,15,20,26 Concurrent underlying
disease processes are also often identified.
Histopathology of affected tissues reveals yeasts, pseudo-
hyphae, and septate hyphae with an associated inflammatory
response that is composed primarily of neutrophils, macro-
phages, and lymphocytes. Multinucleated giant cells may also
be present.15,20,26 Pseudohyphae and hyphae predominate in
invasive infections (see Figure 4-1). Evidence of blood vessel
invasion, thrombosing vasculitis, and infarction may be seen,
with multifocal mycotic granulomas in affected organs.15 The
organism is most readily visualized with special stains such
as periodic acid–Schiff and Gomori’s methenamine silver.
Immunohistochemistry has also been used to identify Candida
within lesions.15,20 When mixed infections with Candida and
other bacteria are present, tissue gram stains may reveal bac-
terial rods and/or cocci in addition to gram-positive fungal
elements.
Treatment and Prognosis
Treatment of candidiasis should involve management of under-
lying immunosuppressive disorders, if possible, and specific
antifungal drug treatment. Systemic antibacterial drugs should
be discontinued if not clearly indicated, or the spectrum of
activity should be narrowed as much as possible. Cutaneous
and corneal infections are usually treated with a combination of
topical antiseptics or antifungal medications and systemic anti-
fungal drugs until lesions resolve.8
Dogs and cats with Candida UTIs should initially be treated
with antifungal drugs that achieve high concentrations in the
urine, such as fluconazole with or without 5-flucytosine. Sys-
temic antifungals alone may be adequate to resolve Candida
UTIs in some animals, although resolution has been reported
in some animals in the absence of antifungal drug therapy.2
Persistent infection may result from drug resistance, adverse
effects of treatment that necessitate treatment discontinuation,
or inadequate drug penetration into balls of fungus that line
the bladder wall (see Case Example). Intravesicular treatment
with 1% clotrimazole may be effective in these cases.30,31 Under
anesthesia, the bladder is catheterized with a Foley catheter,
emptied, then lavaged with sterile saline. The bladder is then
filled with 1% clotrimazole solution (e.g., 30 to 50 mL for a
cat). The animal is rotated to promote contact of the solution
with the uroepithelium, and after the solution has been in the
bladder for an hour, the catheter is removed and the animal is
allowed to recover. Treatment is continued weekly until Can-
dida can no longer be isolated from the urine. Typically one to
four treatments are required.11,30 Ultrasound-guided adminis-
tration of clotrimazole via cystocentesis to a dog with Candida
cystitis was described in one report.31
Successful treatment of systemic candidiasis in dogs or cats is
rarely described. One dog with Candida peritonitis was treated
successfully by surgical exploration and lavage of the abdomen,
placement of an abdominal drain, and intravenous fluconazole.4
In addition, aggressive supportive measures were required that
included intravenous fluid therapy, fresh frozen plasma, antiemet-
ics, gastroprotectants, total parenteral nutrition, and antibacterial
drugs for a suspected catheter-related infection. Antifungal drugs
other than fluconazole used to treat invasive candidiasis in human
patients include amphotericin B or an echinocandin such as
caspofungin,32 both of which must be administered parenterally.
Prevention
Prevention of candidiasis involves avoidance of excessive immu-
nosuppression as well as discriminate use of antibacterial drugs,
which should be tailored to the results of culture and susceptibil-
ity whenever possible. Whether Candida species carried on the
hands of humans can colonize dogs and cats is not known, but
routine hand washing and the wearing of gloves when immu-
nosuppressed animals are handled or intravascular devices are
placed should minimize the chance of health care–associated
infections. Although prophylactic antifungal drug treatment has
been used to prevent invasive candidiasis in human high-risk
groups, the low incidence of candidiasis in immunosuppressed
dogs and cats does not warrant the use of prophylactic antifungal
drugs in this group.
Public Health Aspects
Candida species (especially C. albicans) are common causes
of mucosal disease in humans. Since 2000, Candida has also
emerged as an extremely important cause of invasive infections
owing to the more widespread use of invasive medical devices
and immunosuppressive drugs. In hospitalized human patients,
Candida is reportedly the fourth most common organism iso-
lated from the bloodstream.33 Although many human infec-
tions result from invasion of tissues by commensal yeasts in the
face of immune suppression, horizontal transmission of Can-
dida and hospital-acquired infections have been described.34
Whether Candida isolates from dogs or cats colonize humans
is not known.
 CHAPTER 67  Candidiasis 657

CASE EXAMPLE
Signalment: “Lizzie”, an 18-year-old female spayed domestic
shorthair from the San Francisco Bay area (Figure 67-3, A)
History: Lizzie was evaluated by the University of California,
Davis, Veterinary Medical Teaching Hospital for a 12-hour
history of dysuria and hematuria. The owner described
multiple episodes of urination and defecation outside the
litter box with stranguria, hematuria, and tenesmus. Lizzie was
also observed repeatedly grooming her perineum. The cat had
been diagnosed with diabetes mellitus 10 years previously, and
more recently with feline bronchial disease, a thoracolumbar
(T3 to L3) myelopathy, generalized osteoarthritis, chronic
constipation, bilateral cataracts, chronic kidney disease, and
recurrent bacterial UTIs (Streptococcus bovis group, Escherichia
coli, Enterococcus faecalis, then Aerococcus viridans). She also
had a cardiac murmur secondary to dynamic right ventricular A
outflow tract obstruction. The historical UTIs had been treated
with a variety of antimicrobial drugs selected based on the
results of aerobic bacterial urine culture and susceptibility
(most recently doxycycline, but also amoxicillin-clavulanic
acid or marbofloxacin in the past). Her current medications
consisted of prozinc insulin (3.5 units SC q12h), lactulose
(2 mL PO q12h), a soluble fiber supplement (1 teaspoon with
food twice daily), inhaled fluticasone (110-µg puff q12h),
theophylline (12 mg/kg PO q24h), and doxycycline (5 mg/
kg PO q12h). Her most recent blood work (collected 1 month
previously) showed a creatinine of 1.8 mg/dL (reference range,
1.1-2.2 mg/dL) and a BUN of 34 (reference range, 18-33 mg/
dL), which were similar to values obtained over the course
of the previous 5 years. She was an indoor-only cat and was
fed a commercial cat food with occasional turkey meat from
the delicatessen. Her appetite was good, there had been no
weight loss noted, her water consumption was unchanged,
and there had been no vomiting or diarrhea. She had not
been vaccinated for the past 4 years but was on regular flea
B
preventative medication (nitenpyram as well as fipronil).
Physical Examination: FIGURE 67-3  A, 18-year-old female spayed domestic shorthair with a Candida
Body Weight: 4.1 kg. albicans urinary tract infection, as well as diabetes mellitus, feline bronchial disease,
General: Quiet, alert and responsive, T = 100.5°F (38.1°C), a myelopathy, and generalized osteoarthritis. B, Bladder lumen as viewed by cystos-
copy. The urine contains large amounts of fluffy white debris that was loosely adherent
HR = 170 beats/min, RR = 28 breaths/min, CRT <1 s, pale pink
to the bladder wall.
mucous membranes.
Integument: A dull, slightly unkempt haircoat with mild scale
was noted. and irregular. The urinary bladder was approximately 5 cm
Eyes, Ears, Nose, and Throat: Immature cataracts were in diameter and soft. Severe erythema was noted around
present bilaterally, and all teeth were absent on examination the vulva.
of the oral cavity. No other clinically significant abnormalities Lymph Nodes: All were <1 cm in diameter.
were detected. Laboratory Findings: Urinalysis: SGr 1.030; pH 6.0, 75 mg/dL
Musculoskeletal: BCS was 4/9, and there was mild generalized protein, 1000 mg/dL glucose, no ketones or bilirubin, 250
muscle atrophy. The cat was reluctant to ambulate, and erythrocytes/µL hemoprotein, >100 WBC/HPF, >100 RBC/
appeared painful on manipulation of the elbows, stifles, HPF, few transitional and squamous epithelial cells, many
and coxofemoral joints bilaterally. Crepitus was detected on budding yeast organisms
manipulation of the tarsus. Microbiologic Testing: Aerobic bacterial urine culture: 105
Cardiovascular and Respiratory Systems: A grade II/VI left organisms/mL Candida albicans were cultured.
parasternal heart murmur was auscultated. Pulses were Yeast antifungal drug susceptibility panel: Susceptible to 5-
strong and synchronous. No other abnormalities were flucytosine (0.5 µg/mL), caspofungin (0.25 µg/mL),
detected. f­luconazole (0.25 µg/mL), itraconazole (0.06 µg/mL), vori-
Abdominal Palpation: The abdomen was soft and nonpainful. conazole (0.015 µg/mL). Minimum inhibitory concentrations
Both kidneys were small (approximately 2 cm in diameter) for amphotericin B and posaconazole were 0.5 µg/mL

Continued
658 SECTION 3  Fungal and Algal Diseases
and 0.03 µg/mL, respectively (no interpretative criteria
available).
Diagnosis: Susceptible C. albicans UTI.
Treatment: Lizzie was treated with fluconazole (50 mg PO q12h).
At a recheck examination 10 days later, the owner reported
ongoing pollakiuria and incontinence. A fungal urine culture
again revealed 105 C. albicans/mL with a similar susceptibility
profile. Addition of flucytosine was recommended (250 mg
PO q8h) but was considered unaffordable to the owners.
Treatment was continued for another month with little clinical
improvement and persistence of infection. A blood glucose
curve revealed two-hourly blood glucose concentrations that
were “high”, 470, 368, 300, 383, and 423 mg/dL, respectively,
so the insulin dose was increased to 4 U q12h. Two weeks
later the owner described some improvement in the urinary
incontinence and reduction in stranguria, but again 105
C. albicans/mL was cultured from the urine, and persistent
urine scalding was noted around the perineum and on the
pelvic limbs on physical examination. Body weight was
3.8 kg, and a blood glucose curve revealed two-hourly blood
glucose concentrations that were 457, 301, 209, 227, 300, and
296 mg/dL, respectively.
Topical treatment with clotrimazole was elected. Before the
procedure was performed, a preanesthetic evaluation was
done. A CBC showed a normocytic, normochromic, non-
regenerative anemia (HCT 28.3%; RR, 30%-50%), mature
neutrophilia (15,206 cells/µL; RR, 2000-9000 cells/µL), lym-
phopenia (919 cells/µL; 1000-7000 cells/µL), and monocy-
topenia (33 cells/µL; 50-600 cells/µL). A serum biochemistry
profile was unremarkable with the exceptions of a BUN con-
centration of 49 mg/dL (18-33 mg/dL) and a glucose concen-
tration of 314 mg/dL (63-118 mg/dL). Urinalysis showed a spe-
cific gravity of 1.022; pH 6.0, 25 mg/dL protein, 1000 mg/dL
glucose, 0-2 WBC/HPF, 0-2 RBC/HPF, and moderate numbers
of budding yeast organisms and fungal structures. Thoracic
radiographs revealed mild cardiomegaly and a diffuse, static
bronchial pattern consistent with the previous diagnosis
of feline bronchial disease. Abdominal ultrasound findings
included an enlarged and hyperechoic liver, mild diffuse
thickening of the gastrointestinal mucosa and submucosa,
decreased renal corticomedullary distinction bilaterally with
thickening of the renal cortices, a markedly thickened and
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J Vet Emerg Crit Care (San Antonio). 2010;20:143-147.
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tions in 13 dogs and seven cats: predisposing factors, treatment, and
outcome. J Am Anim Hosp Assoc. 2003;39:263-270.
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irregular urinary bladder wall that contained hypoechoic,
ill-defined nodules, and thick sediment within the urinary
bladder lumen. Cystoscopy revealed a large amount of
white, fluffy debris adherent to an edematous bladder wall
as well as in the lumen (Figure 67-3, B). A 10F Foley cath-
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was flushed and the majority of the debris removed. A total
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bladder, and the solution was allowed to remain in place for
1 hour. The solution was removed and Lizzie was allowed
to recover from anesthesia. Treatment with fluconazole was
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still urinated outside the litter box and intermittent vomiting
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2009;71:669-672.
14. Ochiai K, Valentine BA, Altschul M. Intestinal candidiasis in a dog.
Vet Rec. 2000;146:228-229.
15. Rodriguez F, Fernandez A, Espinosa de los Monteros A, et  al.
Acute disseminated candidiasis in a puppy associated with parvovi-
ral infection. Vet Rec. 1998;142:434-436.
16. Langheinrich KA, Nielsen SW. Histopathology of feline panleuko-
penia: a report of 65 cases. J Am Vet Med Assoc. 1971;158:suppl
2:863+.
17. Matsuda K, Sakaguchi K, Kobayashi S, et al. Systemic candidiasis
and mesenteric mast cell tumor with multiple metastases in a dog.
J Vet Med Sci. 2009;71:229-232.
18. Miller WW, Albert RA. Ocular and systemic candidiasis in a cat.
J Am Anim Hosp Assoc. 1988;24:521-524.
19. Lorenzini R, De Bernardis F. Antemortem diagnosis of an apparent
case of feline candidiasis. Mycopathologia. 1986;93:13-14.
20. Brown MR, Thompson CA, Mohamed FM. Systemic candidia-
sis in an apparently immunocompetent dog. J Vet Diagn Invest.
2005;17:272-276.
21. Ling GV, Norris CR, Franti CE, et al. Interrelations of organism
prevalence, specimen collection method, and host age, sex, and
breed among 8,354 canine urinary tract infections (1969-1995).
J Vet Intern Med. 2001;15:341-347.
22. Norris CR, Williams BJ, Ling GV, et al. Recurrent and persistent
urinary tract infections in dogs: 383 cases (1969-1995). J Am Anim
Hosp Assoc. 2000;36:484-492.
CHAPTER 67  Candidiasis 659
23. Karkowska-Kuleta J, Rapala-Kozik M, Kozik A. Fungi pathogenic
to humans: molecular bases of virulence of Candida albicans, Cryp-
tococcus neoformans and Aspergillus fumigatus. Acta Biochim Pol.
2009;56:211-224.
24. Cheng SC, Joosten LA, Kullberg BJ, et  al. Interplay between
Candida albicans and the mammalian innate host defense. Infect
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25. Holoymoen JI, Bjerkas I, Olberg IH, et  al. Disseminated can-
didiasis (moniliasis) in a dog. A case report. Nord Vet Med.
1982;34:362-367.
26. Skoric M, Fictum P, Slana I, et  al. A case of systemic mycosis in
a Hovawart dog due to Candida albicans. Veterinarni Medicina.
2011;56:260-264.
27. Kuwamura M, Ide M, Yamate J, et  al. Systemic candidiasis in a
dog, developing spondylitis. J Vet Med Sci. 2006;68:1117-1119.
28. Linek J. Mycotic endophthalmitis in a dog caused by Candida albi-
cans. Vet Ophthalmol. 2004;7:159-162.
29. Rosenvinge FS, Dzajic E, Knudsen E, et al. Performance of matrix-
assisted laser desorption-time of flight mass spectrometry for iden-
tification of clinical yeast isolates. Mycoses. 2012.
30. Toll J, Ashe CM, Trepanier LA. Intravesicular administration of
clotrimazole for treatment of candiduria in a cat with diabetes mellitus.
J Am Vet Med Assoc. 2003;223:1156-1158:1129.
31. Forward ZA, Legendre AM, Khalsa HD. Use of intermittent blad-
der infusion with clotrimazole for treatment of candiduria in a dog.
J Am Vet Med Assoc. 2002;220:1496-1498:1474-1495.
32. Azie N, Neofytos D, Pfaller M, et al. The PATH (Prospective Anti-
fungal Therapy) Alliance registry and invasive fungal infections: update
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33. Pappas PG. Invasive candidiasis. Infect Dis Clin North Am.
2006;20:485-506.
34. Ben Abdeljelil J, Saghrouni F, Emira N, et  al. Molecular typ-
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2011;111:1235-1249.
CHAPTER 68
Miscellaneous Fungal Diseases
Amy M. Grooters
I
n contrast to the well-recognized fungal pathogens that
cause endemic mycoses (such as Blastomyces, Histo-
plasma, and Cryptococcus), miscellaneous fungal patho-
gens comprise large groups of fungi with unfamiliar names
that traditionally have caused disease only sporadically. These
opportunistic fungi have low inherent virulence and typically
cause infection only when normal host resistance mechanisms
are compromised. Over the past 30 years, these opportunists
have become increasingly important in human patients because
immunocompromise associated with chemotherapy, organ
transplantation, and HIV infection has become more preva-
lent. Similarly, over the past 5 to 10 years, the frequency with
which opportunistic fungal infections are encountered in small
animal patients has increased significantly in association with
the use of multiagent immunosuppressive therapy (especially
with cyclosporine) to treat immune-mediated disease in dogs.
Thus, veterinarians are increasingly likely to encounter patients
infected by an opportunistic fungal pathogen with which they
are unfamiliar.
Unlike the common endemic fungal pathogens (for which
a definitive diagnosis can often be made simply by visualiz-
ing the unique morphologic features of the organism in cyto-
logic or histologic samples), opportunistic fungi can only be
identified to genus and species level by culture or molecular
methods. However, they can be assigned to categories based
on their morphologic features in tissue, such as pigmentation,
hyphal diameter, and frequency of septation. These catego-
ries include phaeohyphomycosis (pigmented hyphal or yeast
forms), hyalohyphomycosis (nonpigmented hyphal forms),
eumycotic mycetoma (fibrosing granuloma with black or
white tissue grains consisting of aggregates of pigmented or
nonpigmented fungi, respectively), and zygomycosis (wide,
infrequently septate, nonpigmented hyphae associated with
pyogranulomatous and eosinophilic inflammation). Because
of its similarities to pythiosis and lagenidiosis, zygomycosis is
discussed in Chapter 69. Although identification of a specific
pathogen after culture is ideal, classification of opportunistic
mycoses based on the categories just listed is usually sufficient
to allow the clinician to make a reasonable prediction about
clinical course and prognosis and to make appropriate treat-
ment decisions. Because many opportunistic fungi are com-
mon contaminants and can normally be found on skin, nasal
mucosa, and other nonsterile sites, culture- or PCR-based
identification of a potential opportunistic fungal pathogen
from a skin specimen, nasal swab, or exudate should not be
considered evidence of fungal infection unless there is sup-
portive histologic or cytologic evidence of tissue invasion by a
morphologically compatible organism.
660
Overview of Phaeohyphomycoses
Causes: Alternaria, Bipolaris, Cladophialophora, Curvularia,
Exophiala, Fonsecaea, Moniliella, Phialophora, Ramichlo-
ridium, Ulocladium, and Scolecobasidium, among many
others
Affected Hosts: A variety of animal hosts that include dogs,
cats, horses, and humans
Geographic Distribution: Worldwide
Mode of Transmission: Cutaneous inoculation of organisms
from the environment
Major Clinical Signs: Localized cutaneous nodules that may
appear pigmented, often on the nose or digits; occasion-
ally meningoencephalitis, with associated neurologic
signs
Differential Diagnosis: Neoplasia, cryptococcosis, blastomy-
cosis, sporotrichosis, hyalohyphomycosis, zygomycosis,
mycobacterial infections, actinomycosis, nocardiosis
Human Health Significance: Direct transmission from ani-
mals to humans has not been described; humans develop
infections from environmental sources.
Phaeohyphomycosis
Etiology and Epidemiology
The term “phaeohyphomycosis” refers to cutaneous, subcuta-
neous, cerebral, or disseminated infections caused by pigmented
(also known as dematiaceous) fungi that contain melanin in
their cell walls.1 Infection usually results from cutaneous inoc-
ulation. Fungal genera that have been identified as agents of
phaeohyphomycosis in veterinary patients include Alternaria,
­Bipolaris, Cladophialophora, Curvularia, Exophiala, Fonse-
caea, ­Moniliella, ­Phialophora, Ramichloridium, Ulocladium,
and ­Scolecobasidium, among others (Table 68-1).
Clinical Features
The most common clinical manifestations of phaeohyphomycosis
in immunocompetent small animals are lesions associated with
the digits, pinnae, nasal planum, or nasal cavity in cats (Figure
68-1). 2-8 Cutaneous lesions occur less often in dogs.9 In addition,
granulomatous meningoencephalitis caused by pigmented fungi
 CHAPTER 68  Miscellaneous Fungal Diseases 661

TABLE 68-1
Causative Agents and Histologic/Cytologic Characteristics Associated with Miscellaneous Fungal and Pseudofungal Pathogens
Disease Causative Agents Histologic and Cytologic Characteristics
Phaeohyphomycosis Alternaria, Bipolaris, Phialophora, Clado- Pyogranulomatous inflammation associated with pigmented,
phialophora (Cladosporium), Curvu- irregularly septate hyphae or yeast-like cells that may be
laria, Exophiala, Fonsecaea, Moniliella, solitary or may cluster in small groups or chains
Ochroconis, Ramichloridium, others
Hyalohyphomycosis Acremonium, Fusarium, Geotrichum, Geo- Pyogranulomatous inflammation associated with hyphal ele-
smithia, Paecilomyces, Phialosimplex, ments that have nonpigmented (transparent, hyaline) walls;
Pseudallescheria, Sagenomella, Scedo- Phialosimplex may cause yeast-like forms in tissue
sporium, Schizophyllum, others
Mycetoma Curvularia Pyogranulomatous inflammation associated with aggregates
(black-grain) of pigmented fungal organisms (which appear grossly as
pigmented tissue grains)
Mycetoma Pseudallescheria boydii, Acremonium Pyogranulomatous inflammation associated with aggregates
(white-grain) of nonpigmented fungal organisms (which appear grossly
as nonpigmented tissue grains)
Pythiosis Pythium insidiosum Pyogranulomatous and eosinophilic inflammation associ-
ated with broad (2-7 µm), infrequently septate hyphae (see
Chapter 69)
Lagenidiosis Lagenidium Pyogranulomatous and eosinophilic inflammation associated
with broad (4-25 µm), infrequently septate hyphae (see
Chapter 69)
Zygomycosis Conidiobolus spp., Basidiobolus rana- Pyogranulomatous and eosinophilic inflammation associated
rum in dogs; Rhizomucor, Mucor, and with broad (5-20 µm), infrequently septate hyphae with
Cokeromyces in cats thick prominent eosinophilic sleeve (see Chapter 69)
Aspergillosis Aspergillus terreus, A. deflectus, A. flavi- Suppurative to granulomatous inflammation associated with
pes, A. fumigatus, others multiple, nonpigmented, 3- to 6-µm, septate hyphae with
parallel walls and 45-degree angle branching (see Chapter 65)
Candidiasis Candida albicans, other Candida spp. Suppurative inflammation with numerous 2- to 6-µm oval
yeasts, pseudohyphae (chains of oval yeast cells), and true
hyphae (see Chapter 67)

(especially Cladophialophora bantiana) have been described

both in dogs and cats.10-14 In immunocompromised patients, the
most common presentation appears to be multifocal cutaneous
lesions in dogs treated with multiagent immunosuppressive ther-
apy, especially that which includes cyclosporine.15-17
Animals with phaeohyphomycosis typically have cutaneous
nodules or a visible nasal mass. Infected tissues may appear grossly
pigmented, and thus masses may be confused with melanomas.
Phaeohyphomycoses tend to be locally invasive and may extend to
involve regional lymph nodes (Figure 68-2). Systemic dissemina-
tion more often occurs in animals treated with immunosuppres-
sive drugs, but disseminated infections have also been described in
apparently immunocompetent patients.18,19 Animals with central
nervous system (CNS) phaeohyphomycosis may have neurologic
signs such as obtundation, seizures, abnormal placing reactions,
circling, tremors, ataxia, and abnormal cranial nerve function.

FIGURE 68-1  Left distal thoracic limb of an 8-year-old male neutered Burmese cat
with digital phaeohyphomycosis. (Courtesy University of California, Davis, Veterinary Der-
matology Service.)
Diagnosis
Laboratory and Imaging Abnormalities
Results of a CBC, biochemistry panel, and urinalysis in animals
with cutaneous phaeohyphomycoses are typically unremark-
able. Animals with disseminated disease can have abnormalities
662 SECTION 3  Fungal and Algal Diseases

A B

FIGURE 68-2  Popliteal lymphadenopathy and abscessation caused by Curvularia

spp. infection in a 1-year-old female Yorkshire terrier with cutaneous phaeohyphomycosis.
(Courtesy Amy Grooters, Louisiana State University, Baton Rouge, LA.) C

a­ ssociated with underlying immunosuppressive illness or drug

treatment. Imaging with computed tomography (CT) or MRI in
animals with CNS involvement may reveal focal or multifocal
contrast-­enhancing lesions that may be associated with hydro-
cephalus (Figure 68-3).

Cytologic and Histologic Findings

On histologic or cytologic examination, fungi that cause pha-
eohyphomycosis appear as dark-walled, irregularly septate
hyphae or as yeast-like cells, solitary or in small groups or
chains (­Figure 68-4). Pigmentation may not always be appar-
ent on cytologic examination (Figure 68-5). The presence of
melanin in the walls of lightly-pigmented hyphae can be con-
firmed by the examination of unstained sections, by lowering
the microscope condenser during examination, or by utilization
of a Fontana-Masson stain for melanin.
D
Culture
FIGURE 68-3  Axial T1 postcontrast, T2, and FLAIR magnetic resonance images (A,
Organisms that cause phaeohyphomycosis are readily isolated B, and C, respectively) of the brain of a 6-year-old male neutered Border collie mix that
on routine fungal culture of infected tissue biopsies as well as was evaluated for neurologic signs including seizures and progressive obtundation. In the
from fine-needle aspirate specimens from infected lymph nodes. images shown, there is bilateral but asymmetric ventricular dilation that is severe on the
Further identification is based on colony and conidial mor- right and mild to moderate on the left (asymmetric hydrocephalus). Multifocal regions
phology (see also Figure 4-7, B) and PCR and sequencing of of contrast enhancement are also present (A). Some of these regions appeared nodular;
ribosomal RNA genes. Because pigmented fungi are common others had a ring-like appearance. There is also marked contrast enhancement of the ven-
laboratory contaminants and can sometimes be isolated from tricular ependymal lining. The regions of contrast enhancement correlate with regions of
nonsterile sites on healthy animals (such as skin), positive cul- T2 and FLAIR hyperintensity (B and C) and were isointense on precontrast T1-weighted
tures from potentially contaminated sites should only be con- sequences (not shown). Meningoencephalitis caused by Cladophialophora bantiana and
secondary hydrocephalus was diagnosed on brain biopsy and at necropsy (D). Olive-green
sidered significant if accompanied by cytologic or histologic
pigmented masses can be seen on both the left and right sides of the brain. Interestingly,
evidence of fungal infection with a morphologically compatible the dog also had cutaneous protothecosis. (Courtesy University of California, Davis, Veteri-
organism. nary Neurology and Anatomic Pathology Services.)

Treatment and Prognosis

Pigmented fungi are often poorly responsive to medical therapy,
in part because melanin is a virulence factor. Aggressive surgical
resection is the treatment of choice for solitary cutaneous pha-
eohyphomycosis lesions. The surgeon should attempt to obtain
wide margins at the time of the initial surgery. Digit amputation
is usually indicated for lesions of the distal phalanx. Medical
therapy with itraconazole or posaconazole is recommended for
3 to 6 months after surgery, because recurrence of disease at the
surgical site is common.
For nonresectable lesions, treatment with itraconazole
(10 mg/kg/day) often results in partial or complete resolution of
cutaneous lesions, but recurrence is very common, so prolonged
courses (6 to 12 months) should be recommended. Voricon-
azole or posaconazole may be more effective than itraconazole
for the treatment of phaeohyphomycosis but are significantly

FIGURE 68-4  Histopathology showing small groups of pigmented, yeast-like fungal
organisms in tissue from a nasal mass in a cat with phaeohyphomycosis; H&E stain. (Cour-
tesy Amy Grooters, Louisiana State University, Baton Rouge, LA.)
FIGURE 68-5  Impression smear from the brain of the dog in Figure 68-3. Septate,
branching hyphae are present, but pigmentation is not clearly apparent. Wright’s stain,
1000× magnification. (Courtesy University of California, Davis, Veterinary Pathology
Service.)
more expensive. In addition, voriconazole administration is
not recommended in cats because of significant adverse effects
(see Chapter 9). Recently, long-term voriconazole therapy
(7 to 10 mg/kg/day for 10 to 12 months) was used success-
fully to treat intracranial phaeohyphomycosis in one dog10 and
mycotic peritonitis caused by Exophiala in another.20 Early
combination therapy with amphotericin B, flucytosine, and
extended-spectrum azoles has been recommended for treatment
of human patients with CNS infections,21 but flucytosine can be
prohibitively expensive and is generally not tolerated by dogs
(see Chapter 9).
In the author’s experience, cutaneous phaeohyphomycosis
that occurs in dogs receiving immunosuppressive drug therapy
can be resolved in many cases with itraconazole (10 mg/kg/day)
administered for at least 6 months if the immunosuppressive
drug therapy can be tapered quickly. However, dissemination of
disease can also occur, sometimes despite appropriate antifun-
gal therapy. Discontinuation of cyclosporine treatment in these
animals appears to be important for achieving a good outcome.
CHAPTER 68  Miscellaneous Fungal Diseases 663
Public Health Aspects
Phaeohyphomycosis in humans clinically resembles that in dogs
and cats and may include localized cutaneous lesions in immu-
nocompetent hosts; life-threatening CNS infections in immuno-
compromised or apparently immunocompetent hosts (especially
brain abscesses caused by C. bantiana); and, rarely, dissemi-
nated phaeohyphomycosis, primarily in immunocompromised
hosts.21 Infections caused by pigmented fungi are acquired from
the environment. Although there is no evidence of transmission
between mammalian hosts, routine precautions (such as wear-
ing gloves when handling infected tissues or exudate) should be
followed. Precautions should also be taken to prevent needle-
stick injuries if phaeohyphomycosis is suspected.
Overview of Hyalohyphomycoses
Causes: Acremonium, Fusarium, Geotrichum, Paecilomyces,
Pseudallescheria, Sagenomella, Scedosporium, others
Affected Hosts: A variety of animal hosts that include dogs,
cats, and humans
Geographic Distribution: Worldwide
Mode of Transmission: Inhalation or cutaneous inoculation of
organisms in the environment
Major Clinical Signs: Keratitis; cutaneous lesions; osteomyeli-
tis; pneumonia; disseminated disease with involvement of
lymph nodes, kidneys, liver, spleen, bone, intervertebral
discs, and/or CNS
Differential Diagnosis: Neoplasia, aspergillosis, penicilliosis,
phaeohyphomycoses, sporotrichosis, cryptococcosis,
blastomycosis, candidiasis, endemic mycoses, nocardio-
sis, actinomycosis, mycobacteriosis, zygomycosis
Human Health Significance: Direct transmission from ani-
mals to humans has not been described; humans develop
infections from environmental sources.
Hyalohyphomycosis
Etiology and Epidemiology
The term “hyalohyphomycosis” refers to infections caused by
fungi that are nonpigmented (hyaline or transparent) in tissue.
Genera that have been described as agents of hyalohyphomyco-
sis in veterinary patients include Fusarium, Acremonium, Pae-
cilomyces (especially Paecilomyces variotii and ­Paecilomyces
lilacinus), Pseudallescheria boydii (anamorph Scedosporium
apiospermum), Sagenomella, Phialosimplex, Geosmithia, and
Geomyces, among others (see Table 68-1). By convention,
infections caused by Aspergillus and Penicillium species are not
included in the term “hyalohyphomycosis,” because aspergil-
losis and penicilliosis can usually be identified as such based on
their clinicopathologic features. In general, hyalohyphomycosis
occurs more often in dogs than in cats.
Clinical Features
Hyalohyphomycosis ranges from a local disease confined to the
skin (Figure 68-6), nasal mucosa, or cornea to osteomyelitis,
664 SECTION 3  Fungal and Algal Diseases
FIGURE 68-6  Scedosporium prolificans infection in a domestic shorthair cat. There are
multiple draining skin lesions and soft tissue swelling. (Courtesy Spencer Jang, University
of California, Davis.)
pneumonia, and disseminated disease that can involve one or
more organs including the kidneys, bone marrow, lymph nodes,
liver, spleen, bones, intervertebral discs, and CNS, sometimes
with associated fungemia.22-30
Traditionally, the disseminated and corneal forms of hyalo-
hyphomycosis have been most commonly identified. Therefore,
animals with no overt signs of systemic disease that present with
cutaneous or bone lesions that contain nonpigmented hyphae
should still be evaluated for occult lesions in the chest and
abdomen. Like phaeohyphomycosis, hyalohyphomycosis is an
increasingly common cause of multifocal skin lesions in dogs
receiving multiagent immunosuppressive therapy. These dogs
may or may not have accompanying extracutaneous lesions.
Diagnosis
Cytologic and Histologic Findings
Cytologically and histologically, fungi that cause hyalohypho-
mycosis are nonpigmented, frequently septate, branching hyphae
that are often pleomorphic (Figures 68-7 and Figure 4-6). Special
stains such as periodic acid–Schiff and Gomori’s methenamine
silver may be used to identify organisms more readily.
Culture
The fungi that cause hyalohyphomycosis are readily isolated by
routine fungal culture methods from infected tissues as well as
from fine-needle aspirate specimens from infected lymph nodes,
bones, or abdominal organs. Because these fungi are common
laboratory contaminants and can sometimes be isolated from
the skin or hair of healthy animals, positive cultures from non-
sterile sites should be considered significant only if accompa-
nied by cytologic or histologic evidence of fungal infection with
a morphologically compatible organism. Species identification
is based on conidial morphology (Figure 68-8) and may require
PCR and sequencing of ribosomal RNA genes. Species identi-
fication may assist in selection of antifungal drug treatments,
because some species are predictably less susceptible to conven-
tional antifungal drugs (see Treatment and Prognosis). Proper
identification and antifungal drug susceptibility testing are best
performed by laboratories that specialize in mycoses (e.g., the
FIGURE 68-7  Fine needle aspirate cytology from a lytic bone lesion in a 4-year-old
female spayed mixed breed dog with hyalohyphomycosis. Note the septate, nonpig-
mented fungal hyphae present within the macrophage; Wright-Giemsa stain, bar =10 µm.
(Courtesy Kaikhushroo Banajee, Louisiana State University, Baton Rouge, LA.)
FIGURE 68-8  Cytologic appearance of Paecilomyces variotii growing in culture in the
laboratory. Lactophenol cotton blue, 1000× magnification. (Courtesy Spencer Jang, Uni-
versity of California, Davis.)
University of Texas Health Science Center at San Antonio) and
may be useful for selection of treatments for difficult cases. Nev-
ertheless, evidence that supports a correlation between in vitro
susceptibility data and clinical response to treatment is lacking.
Antigen Testing
The Platelia Aspergillus galactomannan antigen ELISA assay is
positive in some, but not all, animals with hyalohyphomyco-
ses such as paecilomycosis. This results from antigenic cross-
reactivity (see Chapter 65).31 In most dogs with disseminated
paecilomycosis that test positive, the magnitude of positive
results is somewhat lower than those in dogs with dissemi-
nated aspergillosis, but occasionally results are strongly posi-
tive (galactomannan indices >6).
Treatment and Prognosis
Treatment of hyalohyphomycosis has traditionally been chal-
lenging because many animals have disseminated disease. Drugs
used most often to treat hyalohyphomycosis in small animals
include itraconazole and amphotericin B, but different fungal
species vary in their susceptibility to antifungal drugs, and some
species, such as Pseudallescheria boydii and Paecilomyces lilaci-
nus, are relatively resistant to conventional antifungal drugs
in  vitro.32,33 The newer triazoles, voriconazole and posacon-
azole, and the echinocandins, such as caspofungin, may be more
active against these fungi than itraconazole but are significantly

more expensive. In contrast, Paecilomyces variotii is usually sus-
ceptible to amphotericin B but is less susceptible to voriconazole.
Although treatment of disseminated hyalohyphomycosis with
antifungal drugs can prolong survival, clinical signs often recur
even if signs initially resolve. Therefore, disseminated hyalohy-
phomycosis generally carries a guarded to poor prognosis.
Cutaneous hyalohyphomycosis that develops in an animal
receiving immunosuppressive drug therapy may respond well to
oral azole antifungal drugs or may rapidly disseminate. Therefore,
fungal skin lesions that develop in immunocompromised patients
should be treated aggressively, and a guarded prognosis should be
offered. This author most often uses itraconazole (10 mg/kg/day)
administered orally for at least 6 months. Other options include
amphotericin B, voriconazole, or posaconazole. Combination
therapies have been recommended for difficult cases in human
patients.21 Discontinuation of cyclosporine and rapid tapering of
other immunosuppressive medications in these patients appears
to be important for achievement of a good outcome.
Public Health Aspects
Hyalohyphomycosis in humans is acquired as a result of infection
by fungi in the environment. Keratitis and nailbed infections (ony-
chomycosis) can occur in immunocompetent humans. Invasive
infections occur in immunocompromised patients such as trans-
plant recipients, patients with AIDS, and leukemic or neutropenic
patients. Although there is no evidence to suggest that transmission
between mammalian hosts is possible, routine precautions (such as
wearing gloves when handling infected tissues or exudate) should
be followed. Precautions should especially be taken to prevent nee-
dle-stick injuries if hyalohyphomycosis is suspected.
Eumycotic Mycetoma
The term “mycetoma” refers to localized mycotic or actino-
mycotic infections of the skin, subcutaneous tissue, muscle, or
CASE EXAMPLE
Signalment: “CJ”, a 1-year-old male intact boxer dog from
central Louisiana
History: CJ was initially evaluated for acute ataxia and abnormal
limb placement. Based on results of CSF analysis as well as MRI
of the brain and spinal cord, inflammatory central nervous
system disease was diagnosed, and treatment with prednisone
(1 mg/kg PO q12h) was initiated. Despite a good initial response,
neurologic signs recurred 2 weeks later, at which time cytosine
arabinoside (50 mg/m2 SC q12h, for four doses, repeated every
3 weeks) and azathioprine (1.6 mg/kg PO q48h) were added
to the treatment protocol. Neurologic signs again improved,
but 7 weeks later, the dog was evaluated for ulcerative lesions
on the distal extremities that had been present for 1 week and
lethargy that had been present for 3 days.
Physical Examination:
Body Weight: 29 kg.
General: Alert and responsive, but painful on ambulation
because of ulcerative lesions on all four feet. Vital parameters
were within normal limits. Peripheral lymph nodes were
palpably normal.
CHAPTER 68  Miscellaneous Fungal Diseases 665
bone that are characterized by the presence of colonies or aggre-
gates of organisms that form “grains” in tissue. Actinomycotic
mycetomas are caused by bacteria such as Actinomyces spp. or
Nocardia spp. (see Chapters 42 and 43), dermatophytic myceto-
mas are caused by dermatophytes (see Chapter 58), and eumy-
cotic mycetomas are caused by nondermatophyte fungi. Lesions
result from traumatic implantation of soil organisms into tissue.
The grains or granules of eumycotic mycetomas are pigmented
(for black-grain mycetoma, caused by dematiaceous fungi) or
hyaline (for white-grain mycetoma, caused by nonpigmented
fungi), depending on the type of fungal pathogen involved (see
Table 68-1).
Black-grain mycetomas are most often caused by Curvu-
laria species and typically are associated with chronic nonheal-
ing wounds and cutaneous nodules on the extremities. Lesions
often develop weeks to months after a traumatic incident in the
same area. Draining tracts are often present, and black grains
may be observed in the exudate. White-grain mycetomas, usu-
ally caused by Pseudallescheria boydii or Acremonium spp.,
most often occur as body-wall and/or intra-abdominal granulo-
mas that develop subsequent to surgical wound contamination
or dehiscence. Interestingly, the lesions associated with white-
grain mycetoma may not be evident until months or even a year
or more after the surgical event. Affected dogs may be evalu-
ated for a draining mass on the body wall or clinical signs of
peritonitis.
The treatment of choice for eumycotic mycetoma is aggres-
sive surgical excision of infected tissues, including amputation
if clinically indicated. Response to medical therapy is gener-
ally poor, but administration of itraconazole, voriconazole, or
posaconazole may be considered as adjunctive therapy follow-
ing surgical excision when wide margins cannot be achieved.
Dissemination of eumycotic mycetoma beyond local tissues
is rare, but disease within the abdomen or other local tissues
may be extensive. The prognosis is guarded if complete surgical
resection is not possible.
Dermatologic Examination: Pitting edema and multifocal
ulcerative lesions were present on all four distal
extremities, with sloughing of multiple footpads (Figure
68-9, A and B). Some of the pad lesions were proliferative
and extended into the interdigital area (Figure 68-9, C).
Multiple light brown colored areas could be visualized
within the fibrinonecrotic material that covered the area
of the left metacarpal pad (see Figure 68-9, B). In addition,
a 1-cm ulcerated lesion was present on the left pinna, a
crusted lesion was present on the tip of the tail, and a small
subcutaneous mass was present in the right saphenous
area. Larger (3 cm) ulcerated lesions partially covered with
eschar were present on the medial aspect of the right
tarsus and the dorsal aspect of the left antebrachium. Major
differential diagnoses considered were opportunistic
fungal infection and drug eruption.
Cytology and Histology Findings: Cytologic evaluation of
the left pinnal lesion, subcutaneous right saphenous mass,
and right popliteal lymph node revealed septic neutrophilic
to pyogranulomatous inflammation with septate,
branching, 3- to 7-µm fungal hyphae, both extracellularly
and within neutrophils. Pigmentation of hyphae was not
observed.
Continued
666 SECTION 3  Fungal and Algal Diseases
Histologic evaluation of punch biopsies obtained from cutane-
Diagnosis: Disseminated cutaneous phaeohyphomycosis
Treatment and Outcome: Pentoxifylline (30 mg/kg PO
A B
Comments: As was noted here, pigmentation of hyphae may
C
FIGURE 68-9  A-C, Phaeohyphomycosis of the left thoracic limb that developed
after multiagent immunosuppressive therapy in 1-year-old boxer with inflammatory
central nervous system disease. (Courtesy Amy Grooters, Louisiana State University, Baton
Rouge, LA.)
SUGGESTED READINGS
Grooters AM, Foil CS. Miscellaneous fungal infections. In: Greene CE,
ed. Infectious Diseases of the Dog and Cat. 4th ed. Philadelphia, PA:
WB Saunders; 2012:675-688.
Naggie S, Perfect JR. Molds: hyalohyphomycosis, phaeohyphomycosis
and zygomycosis. Clin Chest Med. 2009;30:337-353.
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4. Knights CB, Lee K, Rycroft AN, et al. Phaeohyphomycosis caused
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6. Fondati A, Gallo MG, Romano E, et al. A case of feline phaeohypho-
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8. Tennant K, Patterson-Kane J, Boag AK, et al. Nasal mycosis in two
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tion in a dog. J Vet Diagn Invest. 2008;20:379-381.
10. Bentley RT, Faissler D, Sutherland-Smith J. Successful management
of an intracranial phaeohyphomycotic fungal granuloma in a dog.
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11. Giri DK, Sims WP, Sura R, et  al. Cerebral and renal phaeohy-
phomycosis in a dog infected with Bipolaris species. Vet Pathol.
2011;48:754-757.

12. Anor S, Sturges BK, Lafranco L, et al. Systemic phaeohyphomyco-
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13. Bouljihad M, Lindeman CJ, Hayden DW. Pyogranuloma-
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14. Simmons JK, McManamon R, Rech RR, et al. ­Pathology in ­Practice.
Necrotizing pyogranulomatous ­meningoencephalitis with
­intralesional fungal hyphae, consistent with ­Cladophialophora
bantiana. J Am Vet Med Assoc. 2010;236(3):295-297.
15. Dedola C, Stuart AP, Ridyard AE, et  al. Cutaneous Alternaria
infectoria infection in a dog in association with therapeutic immu-
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lytic anaemia. Vet Dermatol. 2010;21:626-634.
16. Swift IM, Griffin A, Shipstone MA. Successful treatment of dis-
seminated cutaneous phaeohyphomycosis in a dog. Aust Vet J.
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17. Herraez P, Rees C, Dunstan R. Invasive phaeohyphomycosis caused
by Curvularia species in a dog. Vet Pathol. 2001;38:456-459.
18. Coldrick O, Brannon CL, Kydd DM, et  al. Fungal pyelone-
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2007;161:724-728.
19. Elies L, Balandraud V, Boulouha L, et al. Fatal systemic phaeohy-
phomycosis in a cat due to Cladophialophora bantiana. J Vet Med
A Physiol Pathol Clin Med. 2003;50(10):50-53.
20. Murphy KF, Malik R, Barnes A, et al. Successful treatment of intra-
abdominal Exophiala dermatitidis infection in a dog. Vet Rec.
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21. Naggie S, Perfect JR. Molds: hyalohyphomycosis, phaeohyphomy-
cosis and zygomycosis. Clin Chest Med. 2009;30:337-353.
22. Foley JE, Norris CR, Jang SS. Paecilomycosis in dogs and horses
and a review of the literature. J Vet Intern Med. 2002;16:238-243.
CHAPTER 68  Miscellaneous Fungal Diseases 667
23. Holahan ML, Loft KE, Swenson CL, et  al. Generalized calcino-
sis cutis associated with disseminated paecilomycosis in a dog. Vet
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24. Caro-Vadillo A, Garcia-Real I, Paya-Vicens MJ, et al. Fungal rhini-
tis caused by Scedosporium apiospermum in a Labrador retriever.
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25. Erne JB, Walker MC, Strik N, et al. Systemic infection with Geo-
myces organisms in a dog with lytic bone lesions. J Am Vet Med
Assoc. 2007;230:537-540.
26. Tanaka H, Takizawa K, Baba O, et  al. Basidiomycosis: Schizo-
phyllum commune osteomyelitis in a dog. J Vet Med Sci.
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27. Grant DC, Sutton DA, Sandberg CA, et al. Disseminated Geosmi-
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28. Armstrong PF, Sigler L, Sutton DA, et al. Fungal myelitis caused by
Phialosimplex caninus in an immunosuppressed dog. Med Mycol.
2012;50:509-512.
29. Marlar AB, Miller PE, Canton DD, et al. Canine keratomycosis: a
report of eight cases and literature review. J Am Anim Hosp Assoc.
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30. Rampazzo A, Kuhnert P, Howard J, et  al. Hormographiella
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32. Lackner M, de Hoog GS, Verweij PE, et al. Species-specific antifun-
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2006;12(10):948-960.
CHAPTER 69
Pythiosis, Lagenidiosis, and Zygomycosis
Amy M. Grooters
P
ythiosis, lagenidiosis, and zygomycosis are often grouped
together because of similarities in their clinical presenta-
tions and histologic characteristics (all three cause pyogran-
ulomatous and eosinophilic inflammation associated with large,
infrequently septate hyphae with nonparallel walls). Despite their
clinicopathologic similarities, however, the pathogens that cause
these infections are taxonomically diverse. Pythium insidiosum
and Lagenidium species are water molds in the class Oomycetes
and as such are more closely related to red algae and Prototheca
spp. than to true fungi, including the zygomycetes.1 Important
traits that distinguish oomycetes from fungi include the produc-
tion of motile, flagellate zoospores that act as infective elements
in wet environments, and the fact that the oomycete cell mem-
brane generally lacks ergosterol.2 In addition, there are clini-
cally relevant differences in prognosis, recommended treatment,
and epidemiology that make it important to distinguish among
pythiosis, lagenidiosis, and zygomycosis. Although P. insidiosum
has been recognized as a pathogen in dogs and horses for more
than 25 years, Lagenidium species have only been recognized as
Overview of Pythiosis
First Described: 1884, India (Smith)3, in horses
Cause: Pythium insidiosum (kingdom Stramenopila, class
Oomycota), related to algae and Prototheca spp.
Affected Hosts: Horses and dogs; less commonly cats, sheep,
cattle, exotic species, and humans
Geographic Distribution: Tropical, subtropical, and some
temperate regions worldwide, especially the Gulf Coast
region of the United States
Mode of Transmission: Likely penetration of damaged skin
or mucosa by motile zoospores, usually after exposure to
standing freshwater sources
Major Clinical Signs: Ulcerative, nodular, and mass-like cuta-
neous lesions with draining tracts (cutaneous form);
weight loss, anorexia, vomiting, diarrhea, hematochezia
(gastrointestinal form)
Differential Diagnosis: For suspected gastrointestinal pythio-
sis, differential diagnoses include neoplasia, zygomycosis,
histoplasmosis, eosinophilic gastroenteritis, or chronic
intestinal foreign bodies; for suspected cutaneous pythio-
sis, they include lagenidiosis, zygomycosis, mycobacterial
infections, actinomycosis, or furunculosis.
Human Health Significance: P. insidiosum is a rare cause of
disease in human patients; infection is not transmitted
directly from affected animals to people.
668
mammalian pathogens since 1999. In comparison to pythiosis
and lagenidiosis, infections caused by the zygomycetes are rare.
PYTHIOSIS
Etiology and Epidemiology
Pythium insidiosum is an aquatic oomycete that causes severe,
progressive gastrointestinal or cutaneous disease that is often
fatal. The infective form of P. insidiosum is thought to be the
motile flagellate zoospore, which is an asexual reproductive
structure produced in wet environments in association with
plant material.4 Zoospores of P. insidiosum are attracted to
damaged tissue, and likely cause infection by encystation on
and invasion of damaged skin or gastrointestinal mucosa.5 In
support of this mechanism of infection, clinical reports have
long suggested an association between pythiosis and frequent
exposure to warm freshwater habitats. However, some infec-
tions occur in dogs with no history of access to lakes or ponds.
Globally, pythiosis occurs in tropical and subtropical cli-
mates, including Southeast Asia, eastern coastal Australia, and
South America (Figure 69-1). In the United States, the disease
occurs most often in the Gulf Coast states, but it has also been
recognized throughout the South; along the East Coast as far
north as Maryland and New Jersey; in the Midwest including
Missouri, Kansas, southern Illinois, and Indiana; and in the
West in Arizona and California.6
Pythiosis is most common in young, large-breed dogs, espe-
cially Labrador retrievers and other outdoor working breeds.
Infected dogs are typically evaluated by veterinarians in the
fall, winter, and early spring.7 Pythiosis is uncommon in cats
compared to dogs, and specific breed and sex predilections have
not been observed in the cases described to date. However,
the development of cutaneous pythiosis in very young animals
(less than 1 year of age) appears to occur more often in cats
than in dogs. For both species, affected animals are typically
immunocompetent.
Clinical Features
The clinical signs associated with pythiosis are caused by gastro-
intestinal or cutaneous lesions, or by extension of disease into
regional lymph nodes or adjacent tissues. Generally, gastrointes-
tinal and cutaneous lesions do not occur together in the same ani-
mal. Disseminated pythiosis has only been described in one dog.8
Gastrointestinal Pythiosis
Gastrointestinal pythiosis in dogs is characterized by severe,
segmental, transmural thickening of the stomach wall (Fig-
ure 69-2), small intestine, colon, rectum, or, rarely, the
 CHAPTER 69  Pythiosis, Lagenidiosis, and Zygomycosis
FIGURE 69-1  Worldwide geographic distribution of pythiosis.
FIGURE 69-2  Gastric pythiosis in a 1-year-old male neutered mixed breed dog. Note
extreme thickening of the gastric wall. (Courtesy Amy Grooters, Louisiana State University,
Baton Rouge, LA.)
esophagus, and it is not uncommon to find multiple segmental
lesions in the same patient.7,9,10 The most commonly affected
regions of the gastrointestinal tract are the gastric outflow
area, proximal duodenum, and ileocolic junction. Mesen-
teric lymphadenopathy is common but is usually caused by
reactive hyperplasia rather than infection. Extension of dis-
ease into the mesenteric root often causes severe mesenteric
lymphadenopathy, with the lymph nodes embedded in a
single large, firm mass that is palpable in the cranial to mid
669
abdomen. Invasion of mesenteric vessels may result in bowel
ischemia, infarction, perforation, or acute hemoabdomen. In
addition, infection may extend into adjacent tissues such as
the pancreas or the uterus. Gastrointestinal pythiosis is rare
in cats but has been described in two young adult male cats
with focal intestinal lesions that were amenable to surgical
resection.11
Dogs with gastrointestinal pythiosis typically have a history
of chronic progressive weight loss, vomiting, diarrhea, anorexia,
and sometimes hematochezia, depending on the location of the
lesions. Regurgitation may be present in dogs with esophageal
involvement. Physical examination usually reveals a poor body
condition and sometimes a palpable abdominal mass. Signs of
systemic illness such as lethargy or depression are absent unless
intestinal obstruction, infarction, or perforation occurs.
Cutaneous Pythiosis
Cutaneous pythiosis in dogs occurs most often at the base of the
tail, or on the extremities, ventral neck, or perineum,8,12 and is
characterized by nonhealing wounds and invasive masses that
contain ulcerated nodules and draining tracts (Figure 69-3). In
contrast to gastrointestinal pythiosis, regional lymphadenopa-
thy associated with cutaneous pythiosis often reflects extension
of infection rather than just reactive hyperplasia. Extension of
cutaneous disease to tissues other than regional lymph nodes
is rare, but the author has observed a single, focal pulmonary
lesion caused by P. insidiosum in one dog with cutaneous
pythiosis on a distal extremity.
In cats, lesions associated with cutaneous pythiosis include
cervical, inguinal, or truncal subcutaneous masses; draining
nodular lesions or ulcerated plaque-like lesions on the tail-
head or extremities12; and periorbital and nasopharyngeal
lesions.13
670 SECTION 3  Fungal and Algal Diseases

Diagnosis
Laboratory Abnormalities
The most common laboratory abnormalities associated with
pythiosis are eosinophilia, anemia, hyperglobulinemia, and
hypoalbuminemia. Hypercalcemia has been described once.14

Imaging Findings
In dogs with gastrointestinal pythiosis, common radiographic
findings include poor abdominal detail and the presence of an
abdominal mass. Evidence of small bowel obstruction may also
be observed. In rare cases of esophageal pythiosis, thoracic
radiographs reveals increased soft tissue opacity in the region
of the esophagus and deviation of the trachea. Abdominal ultra-
sonography is the tool of choice for imaging animals suspected
to have gastrointestinal pythiosis and usually reveals severe seg-
mental thickening of the gastrointestinal tract and mesenteric
lymphadenopathy. In addition, Doppler ultrasonography may
identify the presence of vascular pathology (such as an aneu-
rysm) that can result from invasion of mesenteric vessels. In
the author’s practice, abdominal ultrasonography is a critical
tool for determining the location and extent of disease in dogs
with gastrointestinal pythiosis to determine the likelihood that
surgical resection could be performed successfully and to assess
prognosis. In addition, ultrasonography facilitates the acquisi-
tion of fine-needle aspirate specimens from affected segments of
the gastrointestinal tract and enlarged lymph nodes. In animals
with cutaneous pythiosis, computed tomography may provide
information about the extent of disease and may assist with sur-
gical planning when lesions are not located on an extremity.15

Microbiologic Tests
FIGURE 69-3  Cutaneous pythiosis in a 6-month-old female Labrador retriever.
(Courtesy Amy Grooters, Louisiana State University, Baton Rouge, LA.) Diagnostic assays currently available for pythiosis, lagenidiosis,
and zygomycosis in dogs and cats are described in Table 69-1.

TABLE 69-1
Diagnostic Assays Currently Available for Pythiosis, Lagenidiosis, and Zygomycosis in Dogs and Cats
Assay Specimen Type Target Performance
Cytologic exami- Aspirates of Broad, rarely septate Does not reliably differentiate among pythiosis, lagenidiosis,
nation ­affected tissues fungal hyphae with and zygomycosis.
tapered, rounded ends
Culture Biopsies of affected Pythium insidiosum, Special tissue handling and expertise required for P. insid-
tissues Lagenidium spp., or iosum culture (see text). The use of swab specimens or
zygomycetes aspirates may result in false negatives. Species identifica-
tion requires the use of molecular techniques.
Antibody serol- Serum Antibodies to P. insid- Assays that detect anti-Pythium antibodies allow early, non-
ogy iosum or Lagenidium invasive diagnosis of pythiosis in dogs. Positive test results
spp. correlate with active disease, and titers fall with successful
treatment. Assays that detect Lagenidium antibodies in
dogs are less specific and may be positive in animals with
other diseases.
Histopathology Biopsies of affected Broad, rarely septate Does not reliably differentiate among pythiosis, lagenidi-
tissues fungal hyphae with osis, and zygomycosis. Use of special stains may facilitate
tapered, rounded ends detection.
PCR assays Biopsies of affected P. insidiosum DNA Not widely available.
tissues
 CHAPTER 69  Pythiosis, Lagenidiosis, and Zygomycosis
Cytologic Examination
Cytologically, pythiosis is characterized by pyogranulomatous
and eosinophilic inflammation that can be visualized on impres-
sion smears made from draining skin lesions or fine-needle
aspirates of a thickened gastrointestinal wall or enlarged lymph
nodes. Hyphae are observed occasionally in cytologic samples,
and their morphologic appearance (broad, rarely septate with
tapered, rounded ends) in conjunction with a typical inflam-
matory response can provide a tentative diagnosis of pythiosis,
lagenidiosis, or zygomycosis.14
Culture
Isolation of P. insidiosum from infected tissues is not dif-
ficult but does require specific specimen handling and culture
techniques. For best results, unrefrigerated tissue specimens
should be wrapped in a saline-moistened gauze sponge and
shipped at ambient temperature to arrive within 24 hours
at a laboratory that has experience with culture of patho-
genic oomycetes. Small pieces of fresh, nonmacerated tissue
are placed directly on the surface of vegetable extract agar
supplemented with streptomycin and ampicillin (or an alter-
native selective medium) and incubated at 37°C.16 Mycelial
growth is typically observed within 12 to 24 hours. Isola-
tion of P. insidiosum from swabs of exudate collected from
draining skin lesions is generally unsuccessful. Because gen-
eration of the sexual reproductive structures that are neces-
sary for definitive morphologic classification of pathogenic
oomycetes rarely occurs in the laboratory, identification of
P. insidiosum isolates is based on species-­specific PCR ampli-
fication17 or rRNA gene sequencing. Although production
of zoospores is an important supporting feature for the
identification of pathogenic oomycetes, it is not specific for
P. insidiosum.
Molecular Diagnosis using the Polymerase Chain Reaction
A species-specific PCR assay has been used to identify P. insid-
iosum DNA in fresh, frozen, or paraffin-embedded tissues, as
well as DNA extracted directly from cultured isolates.18 How-
ever, such assays are not widely available on a commercial
basis.
Serologic Testing
A highly sensitive and specific ELISA for the detection of anti–
P. insidiosum antibodies in dogs is currently available through
the author’s laboratory at Louisiana State University.19 This
assay provides a means for early, noninvasive diagnosis and
also allows response to therapy to be monitored. Follow-
ing complete surgical resection of infected tissues, a dramatic
decrease in antibody levels is typically detected within 2 to 3
months. In contrast, antibody levels remain high in animals
that go on to develop clinical recurrence following surgical
treatment. This same ELISA has been adapted for detection of
anti–P. insidiosum antibodies in domestic and exotic cats, and
although case numbers are too small to generate strong esti-
mates of sensitivity and specificity, in the author’s experience
they appear to be high.
Pathologic Findings
Histologically, pythiosis is characterized by eosinophilic pyo-
granulomatous inflammation, with multiple foci of necrosis
that are surrounded and infiltrated by neutrophils, eosino-
phils, and macrophages. In addition, discrete granulomas
671
composed of epithelioid macrophages, plasma cells, multi-
nucleate giant cells, and fewer neutrophils and eosinophils are
often observed. Organisms are typically found within areas
of necrosis or at the center of granulomas. Vasculitis is occa-
sionally present. In gastrointestinal pythiosis, inflammation
centers on the submucosal and muscular layers rather than
the mucosa and lamina propria. Therefore, the diagnosis of
pythiosis may be missed if endoscopic biopsies that fail to
reach deeper tissues are submitted. Similarly, disease in ani-
mals with cutaneous pythiosis is typically found in the deep
dermis and subcutis, necessitating deep wedge biopsies rather
than punch biopsies for optimal evaluation. Pythium insid-
iosum hyphae are not routinely visualized on H&E-stained
sections but may be identified as clear spaces surrounded by
a narrow band of eosinophilic material. Hyphae are readily
visualized in sections stained with Gomori’s methenamine sil-
ver (GMS) but usually do not stain well with periodic acid–
Schiff (PAS). They are wide (mean, 4 µm; range, 2 to 7 µm),
have nonparallel walls, are infrequently septate, and occasion-
ally branch at right angles.7,8
Treatment and Prognosis
Aggressive surgical resection of all infected tissues with wide
margins is the treatment of choice for pythiosis. In animals
with cutaneous lesions that are confined to a single distal
extremity, amputation should be recommended unless there
is evidence of regional lymph node infection. In patients
with gastrointestinal pythiosis, segmental lesions should be
resected with 5-cm margins whenever possible. Because mes-
enteric lymph nodes are usually reactive rather than infected,
the presence of nonresectable mesenteric lymphadenopathy
should not dissuade the surgeon from pursuing resection of
a segmental bowel lesion. Enlarged regional lymph nodes
should, however, always be biopsied for prognostic informa-
tion. In animals with cutaneous pythiosis, the surgeon should
attempt to obtain skin margins of 5 cm and deep margins of
two fascial planes.15 Enlarged regional lymph nodes in dogs
with cutaneous pythiosis are often infected rather than reac-
tive; therefore, they should be evaluated cytologically or his-
tologically before amputation or other aggressive resection is
attempted.
Unfortunately, many dogs with pythiosis are not brought
to a veterinarian until late in the course of disease, when
complete excision is not possible. In addition, the anatomic
location of the lesion may prevent complete surgical excision
when gastrointestinal pythiosis involves the esophagus, gastric
outflow tract, rectum, or mesenteric root, or when cutaneous
pythiosis involves the ventral thorax, base of the tail, or trunk.
Still, very aggressive surgery such as a partial gastrectomy14 or
massive resection of cutaneous lesions combined with recon-
structive techniques15 can sometimes result in a successful
outcome.
Local postoperative recurrence of pythiosis is common
(especially when wide surgical margins cannot be achieved)
and can occur either at the site of resection or in regional
lymph nodes. For this reason, postoperative medical therapy
is often recommended when the surgeon is not confident that
margins of at least 5 cm were achieved. In the author’s prac-
tice, dogs with focal midjejunal lesions that are resected with
wide margins and dogs without regional lymphadenopathy
that undergo amputation for distal extremity lesions are not
672 SECTION 3  Fungal and Algal Diseases
routinely treated with postoperative antifungal medication.
All other patients with pythiosis typically are treated with itra-
conazole (10 mg/kg PO q24h) and terbinafine (5 to 10 mg/kg
PO q24h) for at least 2 to 3 months after surgical resection.
Unfortunately, if resection is incomplete, lesions often progress
despite medical therapy, and clinical signs recur within weeks
to months.
To monitor for recurrence, ELISA serology should be
performed at the time of surgery and 2 to 3 months later. In
animals that have had a complete surgical resection and subse-
quently show no recurrence of disease, serum antibody levels
usually drop 50% or more within 3 months. If this occurs,
medical therapy can be discontinued. In the author’s experi-
ence, surgery is curative in a majority of animals with a dis-
tal limb lesion treated with amputation, or with a midjejunal
lesion that the surgeon believes was completely resected with
good margins.
Medical therapy for nonresectable pythiosis is typically
unrewarding, likely because ergosterol (the target for most
currently available antifungal drugs) is generally lacking in the
oomycete cell membrane. Despite this fact, clinical and sero-
logic cures occur in some patients treated with a combination
of itraconazole (10 mg/kg PO q24h) and terbinafine (5 to 10
mg/kg PO q24h).20 Although the percentage of animals that
respond to this treatment is still low, the combination protocol
anecdotally appears to be superior to itraconazole or ampho-
tericin B alone.
Dogs with nonresectable gastrointestinal pythiosis are often
treated with anti-inflammatory doses of glucocorticoids in an
effort to palliate clinical signs and to decrease vomiting so that
oral antifungal drugs can be administered. Prednisone (1 mg/
kg/day) routinely causes improvement in clinical signs in the
short term. Surprisingly, the author has observed complete and
long-term resolution of gastrointestinal lesions (based on serial
ultrasonographic examinations and serologic testing) in a small
number of dogs treated with prednisone alone. Although this is
certainly not recommended as a primary treatment for animals
with resectable lesions, it is a reasonable option in animals with
nonresectable gastrointestinal lesions, especially when financial
concerns preclude the use of antifungal medication. The author
has not observed this effect of prednisone in dogs with cutaneous
pythiosis.
An immunotherapy product derived from antigens of
P. insidiosum has been used successfully to treat pythiosis in
horses and people.21,22 Unfortunately, although controlled tri-
als have not been completed, the efficacy of this product in dogs
appears to be poor, and clinical improvement has not been
observed in any of the author’s patients.
Public Health Aspects
Human infections caused by P. insidiosum are rare. Infections
are acquired from the environment and have resulted in kerati-
tis, periorbital cellulitis, and arteritis of the lower extremities.
Most cases have been reported from Thailand, and almost all
patients have thalassemia. Extremely rare reports of the disease
exist from humans in North, South, and Central America, Aus-
tralia, and other parts of Asia, many of whom are apparently
healthy. Although there is no evidence to suggest that transmis-
sion between mammalian hosts is possible, routine precautions
(such as wearing gloves when handling infected tissues or exu-
date) should be followed.
Overview of Lagenidiosis
First Described: 2003, Louisiana, USA, in dogs (Grooters and
others) 23
Cause: Lagenidium spp.
Affected Hosts: Dogs
Geographic Distribution: Southeastern United States
Mode of Transmission: Likely penetration of damaged skin or
mucosa by motile zoospores
Major Clinical Signs: Ulcerative, nodular, and mass-like cuta-
neous lesions with draining tracts (cutaneous form);
pelvic limb edema; local, thoracic, or abdominal lymph-
adenopathy; rupture of infected great vessels in the abdo-
men may result in hemoabdomen
Differential Diagnosis: Neoplasia, pythiosis, zygomycosis,
mycobacterial infections, actinomycosis, snake bite
Human Health Significance: Lagenidium spp. infection is
acquired from the environment; infection is not transmit-
ted from affected animals to people.
LAGENIDIOSIS
Etiology and Epidemiology
Most species in the genus Lagenidium are pathogens of insects,
crustaceans, algae, and nematodes. The most well studied species,
Lagenidium giganteum, is a mosquito larval pathogen that was
previously used as a biologic pesticide for control of mosquito
populations. Within the past 15 years, two novel oomycetes that
appear to belong to the genus Lagenidium have been isolated
from dogs with cutaneous lesions. The first of these species causes
uniformly fatal dermatologic and disseminated disease in dogs in
the southeastern United States.23 The second pathogenic Lagen-
idium species is less common than the first and causes a chronic
ulcerative nodular dermatopathy that has a prolonged course and
does not appear to extend beyond local tissues. Although strong
antigenic and molecular similarities suggest that the first canine
pathogen is closely related to L. giganteum, differences in their
molecular and in vitro growth characteristics suggest that they are
likely distinct species. Sporulation and infectivity for these patho-
gens is thought to be similar to that associated with P. insidiosum
and L. giganteum, causing infection through production of motile
aquatic zoospores that adhere to and encyst in damaged tissues.
The epidemiologic features of lagenidiosis are similar in many
respects to those associated with cutaneous pythiosis. Affected
animals are typically young to middle-aged dogs that live in the
southeastern United States. Although most affected dogs have
lived in Florida or Louisiana, cases in Texas, Tennessee, Ala-
bama, Georgia, South Carolina, Maryland, Virginia, Indiana,
and Illinois have also been identified. A number of infected dogs
have had frequent exposure to lakes or ponds. None have had
historical evidence of immunocompromise or were treated with
immunosuppressive therapy before development of the infection.
Clinical Features
Dogs with Lagenidium infection are typically evaluated for pro-
gressive, multifocal or focal, cutaneous or subcutaneous lesions
 CHAPTER 69  Pythiosis, Lagenidiosis, and Zygomycosis
FIGURE 69-4  Tailhead mass caused by lagenidiosis in a 9-year-old female spayed
mixed breed dog. Note the extensive tissue necrosis. (Courtesy Amy Grooters, Louisiana
State University, Baton Rouge, LA.)
that involve the extremities, mammary region, perineum, or
trunk.23 These lesions may appear as firm dermal or subcuta-
neous nodules, or as ulcerated, thickened, edematous areas of
deep cellulitis with regions of necrosis and numerous draining
tracts (Figure 69-4). As with cutaneous pythiosis, skin lesions in
dogs with lagenidiosis tend to be progressive, locally invasive,
and poorly responsive to medical therapy.
In dogs infected with the more aggressive species of Lagen-
idium, regional lymphadenopathy is often noted and may
occur in the absence of obvious cutaneous lesions. Unlike dogs
with cutaneous pythiosis, these dogs typically have occult
lesions in the thorax or abdomen, which may involve the
great vessels, sublumbar and/or inguinal lymph nodes, lung,
pulmonary hilus, and cranial mediastinum. Animals with
great vessel or sublumbar lymph node involvement usually
have cutaneous or subcutaneous lesions on the pelvic limbs
and often develop pelvic limb edema. Sudden death caused by
great vessel rupture and associated hemoabdomen may occur
in these animals.
In dogs infected with the less aggressive species of Lagen-
idium, lesions tend to be locally invasive but rarely extend
beyond cutaneous and subcutaneous tissues. Distant lesions in
the thorax and abdomen have not been identified, and the clini-
cal course appears to be chronic and slowly progressive; some
dogs have lesions that are somewhat stable for several years.
Diagnosis
Because of its clinical, epidemiologic, and histologic similari-
ties to pythiosis, lagenidiosis is often misdiagnosed as pythio-
sis during routine histologic evaluation. In addition, because
many pathologists are more familiar with the zygomycetes than
the oomycetes, lagenidiosis may also be misdiagnosed as zygo-
mycosis. Although there are subtle differences in hyphal size,
morphology, and distribution among these three infections, his-
tologic lesions associated with lagenidiosis are still often labeled
as “suspected pythiosis” or “suspected zygomycosis” during
routine evaluation. For this reason, clinicians should always
attempt to confirm a suspected histologic diagnosis with serol-
ogy, culture, or molecular diagnostic assays.
673
Laboratory Abnormalities
Laboratory abnormalities are often absent in dogs with lagen-
idiosis; when present, they most often include hyperglobulin-
emia and eosinophilia. Hypercalcemia has been associated with
Lagenidium infection in one dog with cutaneous lesions on all
four limbs and severe infection and enlargement of popliteal,
inguinal, and superficial cervical lymph nodes.
Imaging Findings
Radiographic imaging of the thorax and sonographic imaging
of the abdomen are essential parts of the diagnostic evaluation
in dogs suspected to have lagenidiosis because of the potential
for occult lesions in the thorax or abdomen. These may include
solitary pulmonary nodules; sublumbar, inguinal, and medial
iliac lymphadenopathy; thickening and invasion of the wall
of the aorta or caudal vena cava (sometimes with associated
aneurysm); and retroperitoneal or epaxial masses. Thoracic and
abdominal lesions have not been observed in dogs infected with
the less aggressive Lagenidium species.
Microbiologic Tests
Cytologic Examination
Cytologic examination of lymph node aspirates or impression
smears made from draining lesions in dogs with lagenidiosis
reveal pyogranulomatous to eosinophilic inflammation with or
without broad, poorly septate hyphae.
Culture
The diagnosis of lagenidiosis is best made by culture followed by
PCR and rRNA gene sequencing, which not only provides a defini-
tive diagnosis but is also the only tool currently available that differ-
entiates between the two pathogenic species. Isolation techniques
for Lagenidium spp. are similar to those described for P. insidio-
sum, but with peptone-yeast-glucose (PYG) agar. For best results,
small pieces of fresh, nonmacerated tissue are placed directly on
the surface of the agar and incubated at 37°C. Growth is typically
observed within 24 to 48 hours. Because production of the sexual
reproductive structures necessary for morphologic classification
has not yet been possible, definitive identification of Lagenidium
species is currently based on rRNA gene sequencing. Although
genus-specific PCR assays have been used to identify Lagenidium
DNA extracted from cultured isolates or from infected tissue speci-
mens,18 the clinical importance of differentiating between the two
pathogenic species in regard to prognosis and treatment makes cul-
ture and rRNA gene sequencing the preferred technique.
Serologic Testing
In dogs with supportive clinical signs and histologic findings, immu-
noblot serology for the detection of anti-Lagenidium antibodies
in canine serum can suggest lagenidiosis but must be interpreted
in conjunction with results of serologic testing for P. insidiosum
infection because of the potential for cross-reactivity in serum from
dogs with pythiosis. In addition, the author has observed nonspe-
cific anti-Lagenidium seroreactivity in dogs with other fungal or
nonfungal infections. Therefore, based on currently available data,
serology alone should not be used as a basis for the diagnosis of
lagenidiosis. Serology is not helpful for differentiating between
infection by the two canine pathogenic species of Lagenidium.
Pathologic Findings
Histologically, lagenidiosis shares many characteristics with
pythiosis and zygomycosis, including pyogranulomatous and
674 SECTION 3  Fungal and Algal Diseases
FIGURE 69-5  Broad, thick-walled infrequently septate hyphae associated with gran-
ulomatous vasculitis in a lymph node from a dog infected with the more aggressive species
of Lagenidium. (Courtesy Amy Grooters, Louisiana State University, Baton Rouge, LA.)
eosinophilic inflammation associated with broad, irregularly
branching, infrequently septate hyphae with nonparallel walls.
Multinucleated giant cells and plasma cells are commonly
present. In contrast to P. insidiosum, Lagenidium spp. hyphae
are often visible on H&E-stained sections. On GMS-stained
sections, numerous broad, thick-walled, irregularly sep-
tate hyphae are easily recognized (Figure 69-5). Lagenidium
hyphae typically demonstrate a great deal of variability in size,
but in general are larger than P. insidiosum hyphae, ranging
from 7 to 25 µm in diameter, with an average of 12 µm for
the more aggressive pathogen and 7.5 µm for the less aggres-
sive pathogen. In some sections, hyphae appear as round or
bulbous structures, and right-angle branching is occasionally
observed. A scant to thin eosinophilic sleeve may be noted
around the hyphae.
Treatment and Prognosis
As with pythiosis, aggressive surgical resection of infected tis-
sues is the treatment of choice for lagenidiosis when disease is
confined to a resectable cutaneous lesion. Unfortunately, the
vast majority of dogs infected with the more common (and more
aggressive) Lagenidium pathogen have nonresectable disease
in the thorax, abdomen, or regional lymph nodes by the time
the initial diagnosis is made, and in the author’s experience, the
disease is routinely fatal. In dogs infected with the less aggres-
sive species, surgery that achieves 3- to 5-cm margins is often
curative. Because the species of Lagenidium present is often not
known early in the course of diagnostic evaluation, any dog sus-
pected of having lagenidiosis should be evaluated with thoracic
radiography and abdominal ultrasonography before surgical
resection of cutaneous lesions is attempted. As with pythiosis,
medical therapy for lagenidiosis is usually ineffective. How-
ever, treatment with a combination of itraconazole (10 mg/kg
PO q24h) and terbinafine (5 to 10 mg/kg PO q24h) along with
repeated aggressive surgical resection was curative in one dog
with recurrent multifocal cutaneous lesions caused by the less
aggressive pathogen.
Public Health Aspects
Infections caused by Lagenidium species are acquired from
the environment. Although there is no evidence to suggest that
transmission between mammalian hosts is possible, routine pre-
cautions (such as wearing gloves when handling infected tissues
or exudate) should be followed.
Overview of Zygomycosis
(Entomophthoramycosis and Mucormycosis)
First Described: Reports of zygomycosis in humans date back
to the 1800s.
Causes: Basidiobolus and Conidiobolus spp. (kingdom Fungi,
order Entomophthorales); Rhizopus, Absidia, Mucor spp.,
and others (kingdom Fungi, order Mucorales)
Affected Hosts: Dogs, cats, humans, horses, sheep, other
mammalian species
Geographic Distribution: Worldwide
Mode of Transmission: Inhalation, ingestion, or cutaneous
exposure to organisms in soil and decaying organic matter
Major Clinical Signs: Entomophthoramycoses are associated
with chronic, slowly progressive subcutaneous, retrobul-
bar, or nasal infections in immunocompetent animals;
mucormycoses are associated with systemic (respiratory
or gastrointestinal) or cutaneous-subcutaneous infec-
tions in immunocompromised animals.
Differential Diagnosis: Neoplasia, pythiosis, lagenidiosis,
other deep mycoses (especially blastomycosis), mycobac-
terial infections, actinomycosis
Human Health Significance: Disease in humans results from
exposure to organisms in the environment, and direct
transmission between animals has not been reported.
ZYGOMYCOSIS
Etiology and Epidemiology
The term “zygomycosis” refers to infections caused by fungi
in the class Zygomycetes, which includes pathogenic fungi of
the genera Basidiobolus and Conidiobolus (order Entomoph-
thorales) and the genera Rhizopus, Absidia, Mucor, and oth-
ers (order Mucorales). In human and veterinary patients, the
Entomophthorales typically cause chronic localized infections
in subcutaneous tissue or nasal submucosa of immunocom-
petent patients that usually reside in tropical or subtropical
locations (entomophthoramycoses), whereas the Mucorales
tend to cause acute, rapidly progressive disease in debilitated
or immunocompromised individuals worldwide (mucormyco-
ses). Basidiobolus spp. and Conidiobolus spp. cause cutane-
ous lesions in dogs that are grossly and histologically similar
to those caused by P. insidiosum and Lagenidium spp. Cul-
ture-confirmed infections caused by pathogens in the order
Mucorales have not been well documented in dogs but have
been described in a small number of cats. Unfortunately, clini-
cal and pathologic information about zygomycosis in small
 CHAPTER 69  Pythiosis, Lagenidiosis, and Zygomycosis
A B
FIGURE 69-6  A, Nasopharyngeal Conidiobolus spp. infection in a 4-year-old male neutered German shepherd dog. B, Note the severe swelling of the nose and muzzle and ulceration
of the nasal planum. (Courtesy Amy Grooters, Louisiana State University, Baton Rouge, LA.)
animal patients is lacking because disease is uncommon to
rare, and establishment of a definitive diagnosis has histori-
cally required tissue biopsy and culture. As a result, small ani-
mal patients with zygomycosis often go undifferentiated from
those with pythiosis.
Basidiobolus and Conidiobolus species are saprophytes
commonly found in soil and decaying plant matter. Cutaneous
infection with Basidiobolus or Conidiobolus likely occurs by
percutaneous inoculation of spores via minor trauma or insect
bites. Infection may also result from inhalation or ingestion of
spores.
Clinical Features
In dogs, humans, horses, sheep, and other mammalian spe-
cies, conidiobolomycosis occurs most often as a nasopha-
ryngeal infection with or without local dissemination into
tissues of the face, retropharyngeal region, and retrobulbar
space. Manifestations of infection in dogs may include nasal
or facial swelling or deformity, nasal discharge, ulceration
of the nasal planum or hard palate, exophthalmos, chemo-
sis, ocular discharge, and sometimes skin lesions near the
eye (Figure 69-6). In animals with retrobulbar disease that
extends into the brain, neurologic signs may occur. Conid-
iobolus infection has been described in a single dog as a cause
of multifocal nodular draining subcutaneous lesions and
regional lymphadenopathy24 and as a cause of pneumonia in
a dog that was receiving chemotherapy.25 Basidiobolomyco-
sis is a rare cause of ulcerative skin lesions in dogs and has
also been reported as a cause of respiratory disease in a dog.26
Disseminated Basidiobolus infection involving the gastroin-
testinal tract and other abdominal organs has been described
in two dogs.7,27
675
In cats, culture-confirmed cases of zygomycosis are sparse
and have been limited to single reports of cats infected with
fungi in the order Mucorales. Rhizomucor was identified as
the cause of duodenal perforation in a 7-month-old cat,28 and
Cokeromyces recurvatus was isolated from abdominal fluid in
a 16-year-old cat following jejunal perforation caused by lym-
phosarcoma.29 In addition, a subcutaneous mass caused by a
Mucor species on the dorsum of the nose of a 14-year-old cat
was treated successfully with posaconazole.30
Diagnosis
Because of their histologic similarities, zygomycosis is often
confused with pythiosis (which is much more common) when
tissue biopsies are evaluated. Unfortunately, there are no sero-
logic, immunohistochemical, or molecular techniques that are
routinely available for the diagnosis of zygomycosis, making
identification of infected animals reliant on fungal culture of
fresh tissues. As a result, a definitive diagnosis of zygomycosis
is often elusive.
Laboratory and Imaging Abnormalities
Laboratory abnormalities are not well described in animals
with zygomycosis. Diagnostic imaging in the form of computed
tomography often provides important information about the
location and extent of disease in animals with nasopharyngeal
and retrobulbar lesions caused by Conidiobolus spp. In addi-
tion, ocular ultrasonography may help to characterize the extent
of disease in dogs with retrobulbar lesions.
Cytologic and Histologic Findings
The cytologic and histologic features of zygomycosis are simi-
lar to those associated with pythiosis and lagenidiosis. On
676 SECTION 3  Fungal and Algal Diseases
FIGURE 69-7  Conidiobolus spp. hyphae in a tissue biopsy from an ulcerated palate
lesion in a 3-year-old female boxer. Note the large amount of amorphous eosinophilic
material (sleeve) surrounding the hyphal segment; H&E stain. (Courtesy Amy Grooters,
Louisiana State University, Baton Rouge, LA.)
GMS-stained sections, hyphae are broad, thin-walled, and
occasionally septate. The histologic hallmark of zygomycosis
is the presence of a wide (2.5 to 25 µm) eosinophilic sleeve that
surrounds the hyphae and makes them easily located on H&E-
stained sections (Figure 69-7). This finding helps to differen-
tiate zygomycosis from pythiosis and lagenidiosis, in which
eosinophilic sleeves tend to be thin or absent. The hyphal diam-
eter is also significantly larger for Basidiobolus spp. (mean 9
µm; range, 5-20 µm) and Conidiobolus spp. (mean 8 µm; range,
5-13 µm) than for P. insidiosum (mean, 4 µm; range, 2-7 µm).31
CASE EXAMPLE
Signalment: “Jasper,” an 18-month-old intact male Labrador
retriever from central Louisiana
History: Jasper was evaluated for a 1-month history of
frequent vomiting and persistent diarrhea and a 2-week
history of intermittent anorexia. In addition, the owner
reported that the dog had lost approximately 6 kg. The
diarrhea was watery and yellowish-brown and was produced
in large quantity; hematochezia had not been noted. A fecal
flotation and heartworm antigen test performed by the
referring veterinarian 1 week before the dog was evaluated
at the author’s institution had been negative. Treatment
with metronidazole (17 mg/kg PO q12h) and a change to
an easily digestible prescription diet had failed to improve
the vomiting and diarrhea. The patient was a field trial dog
used for duck and dove hunting in central Louisiana. He
frequently worked in rice fields and occasionally in bodies
of fresh water and was generally housed in an outdoor
kennel.
Physical Examination:
Body Weight: 30 kg.
General: Bright, alert, and responsive. Body temperature =
102.7°F (39.2°C), HR = 110 beats/min, RR = 20 breaths/min,
mucous membranes pink, CRT ~ 2 s.
Eyes, ears, nose, and throat: No significant abnormalities
were noted.
Treatment and Prognosis
Recommendations for the treatment of zygomycosis are not
straightforward because attempted therapy has only been
described in a few patients with culture-confirmed diagnoses.
Although anecdotal information as well as a small number of
cases in the literature suggest that cutaneous zygomycosis may
be less aggressive than cutaneous pythiosis or lagenidiosis, pro-
gression of lesions and sometimes even dissemination despite
treatment have also been observed in zygomycete-infected dogs.
The author’s current recommendation for dogs with nasopha-
ryngeal conidiobolomycosis is treatment with itraconazole
(10 mg/kg PO q24h) for at least 6 months. Recurrence is com-
mon after medication is discontinued, so a prolonged course
is usually prescribed. Newer oral azoles such as posaconazole
would also be reasonable choices but are significantly more
expensive. Cutaneous zygomycosis in small animal patients
should be treated with aggressive surgical resection of infected
tissues whenever possible, followed by itraconazole therapy
for 2 to 3 months. If resection is not possible, treatment with
itraconazole, posaconazole, or lipid-complexed amphotericin B
should be recommended.
Public Health Aspects
Zygomycosis in human patients is rare. Infections are acquired
from the environment. Although there is no evidence to suggest
that transmission between mammalian hosts occurs, routine
precautions (such as wearing gloves when handling infected tis-
sues or exudate) should be followed.
Musculoskeletal: The dog was underweight with a body
condition score of 3/9.
Cardiovascular and Respiratory: Strong femoral pulses. No
murmurs or arrhythmias were auscultated. Respiratory rate
and effort were normal.
Gastrointestinal and Genitourinary: The patient showed
signs of discomfort during abdominal palpation; a large,
firm, tubular mass was palpated in the midabdomen. Mild
prostatomegaly was detected on rectal examination.
Lymph nodes: All palpable peripheral lymph nodes were
normal in size and shape.
Laboratory Findings:
CBC:
HCT 33.7% (37%-55%)
MCV 61.7 fL (62-77 fL)
MCHC 20.8 g/dL (32-37 g/dL)
WBC 10,600 cells/µL (8000-14,500 cells/µL)
Neutrophils 6200 cells/µL (3000-11,500 cells/µL)
Lymphocytes 1700 cells/µL (1000-4800 cells/µL)
Monocytes 1100 cells/µL (100-1400 cells/µL)
Platelets 254,000/µL (220,000-600,000 platelets/µL).
Serum Chemistry Profile:
Sodium 142 mmol/L (140-153 mmol/L)
Potassium 4.4 mmol/L (3.8-5.5 mmol/L)
Chloride 110 mmol/L (107-115 mmol/L)
Bicarbonate 20 mmol/L (17-27 mmol/L)
Phosphorus 4.6 mg/dL (3.4-6.3 mg/dL)
Calcium 9.1 mg/dL (9.4-11.4 mg/dL)
 CHAPTER 69  Pythiosis, Lagenidiosis, and Zygomycosis 677

BUN 11 mg/dL ( 8-22 mg/dL)

Creatinine 0.7 mg/dL (0.5-1.7 mg/dL)
Glucose 96 mg/dL (80-115 mg/dL) Jejunum
Total protein 5.7 g/dL (5.8-7.5 g/dL)
Albumin 2.0 g/dL (2.6-4.2 g/dL)
Globulin 3.7 g/dL (2.5-4.0 g/dL)
ALT 34 U/L (0-60 U/L)
AST 22 U/L (0-50 U/L)
ALP 13 U/L (0-100 U/L)
Creatine kinase 134 U/L (0-200 U/L)
Cholesterol 158 mg/dL (150-240 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.4 mg/dL).
Urinalysis: SGr 1.041, pH 7.5, 3+ protein, 3+ bilirubin, negative
hemoglobin, negative glucose, negative ketones, 0-5 WBC/
HPF, 0 RBC/HPF, many lipid droplets.
Imaging Findings:
Plain Abdominal Radiographs: There was a generalized
A Dist 1.16 cm
loss of serosal detail in the abdomen. Gas was present in
the gastrointestinal tract, but there was no evidence of
abnormal intestinal tract dilation.
Abdominal Ultrasound: A severely thickened segment of Mes L Node
small bowel could be visualized extending from the cranial
to the caudal abdomen. The wall of this bowel segment
measured 1.1 cm in thickness and lacked normal layering
(Figure 69-8, A). Mesenteric lymph nodes were enlarged and
heterogenous (Figure 69-8, B). There was a small amount of
free abdominal fluid.
Cytology Findings: Cytologic evaluation of ultrasound-
guided mesenteric lymph node aspirates revealed reactive
lymphoid hyperplasia. Examination of ultrasound-guided
aspirates of the thickened segment of bowel showed that
the cellularity was too low for cytologic evaluation.
Serology Findings: ELISA serology for anti–Pythium Dist 4.11 cm
Dist 1.79 cm
insidiosum antibodies: Positive at 69%. Results of this assay are
expressed as percent positivity in comparison with a strong
B
positive control sample, with values > 40% positivity having
been shown to be 100% specific for pythiosis in dogs. Values FIGURE 69-8  Sonographic images of the abdomen of the dog described in the
in healthy dogs range from 3% to 15%.19 case example. Note the severe thickening and loss of layering in the wall of the jejunum
Diagnosis: Jejunal pythiosis. (A) and the enlarged, heterogenous appearance of the mesenteric lymph node (B).
Treatment: Abdominal exploratory revealed a 50-cm segment
of the mid-jejunum that was severely thickened and firm.
This segment was resected with 5-cm margins. After an and had gained 3 kg body weight. Reevaluation of anti–
uneventful postoperative recovery, the dog’s vomiting, Pythium insidiosum antibody serology at that time yielded
diarrhea, and anorexia resolved quickly. He was treated a percent positivity of 20%. Because the rapid decrease in
with itraconazole (10 mg/kg PO q24h) and terbinafine (8.3 antibody levels from 69% to 20% was strongly suggestive
mg/kg PO q24h) for 2 months in an effort to decrease the of a surgical cure of gastrointestinal pythiosis, oral
chance of recurrence of disease at the site of resection. Liver antifungal medications were discontinued. Reevaluation
enzyme activities and serum bilirubin concentration were of anti–Pythium insidiosum antibody serology 2 years
monitored during this time and remained within reference later yielded results within the range observed in healthy
intervals. dogs (6%).
Histopathologic Findings: Both the resected segment of Comments: Although most dogs with gastrointestinal
jejunum as well as jejunal and ileocolic lymph nodes were pythiosis have nonresectable lesions, those with mid-
submitted for histologic examination. Results revealed jejunal lesions can often be surgically cured. Preoperative
severe transmural eosinophilic pyogranulomatous enteritis abdominal ultrasound is essential since it helps to
and lymphoid hyperplasia. In the jejunal lesion, wide (3- to determine whether or not a gastrointestinal lesion might be
8-µm), poorly septate hyphae with nonparallel walls were surgically resectable. Because medical therapy alone is often
visualized on GMS-stained sections. No organisms were unsuccessful for the treatment of gastrointestinal pythiosis,
observed in the lymph node sections. surgery should be pursued unless there is clear evidence
Follow-up: At reevaluation 2 months after resection of the that lesions are nonresectable. Postoperative medical
jejunal lesion, the dog remained free of clinical signs therapy was probably not necessary in the case described

Continued
678 SECTION 3  Fungal and Algal Diseases
here, since the postoperative clinical course and follow-up
serology results strongly supported a surgical cure. In the
author’s experience, most dogs with a midjejunal lesion are
surgically cured when the surgeon is confident that greater
than 5-cm margins of healthy tissue were obtained with
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16. Grooters AM, Whittington A, Lopez MK, et  al. Evaluation of
microbial culture techniques for the isolation of Pythium insidio-
sum from equine tissues. J Vet Diagn Invest. 2002;14:288-294.
the resection and lymph node histology shows hyperplasia
rather than infection. In such cases, medical treatment with
itraconazole and terbinafine can be offered as optional but
likely unnecessary therapy for 2 months until follow-up
serology is performed.
17. Grooters AM, Gee MK. Development of a nested PCR assay for
the detection and identification of Pythium insidiosum. J Vet Intern
Med. 2002;16:147-152.
18. Znajda NR, Grooters AM, Marsella R. PCR-based detection of
Pythium and Lagenidium DNA in frozen and ethanol-fixed animal
tissues. Vet Dermatol. 2002;13:187-194.
19. Grooters AM, Leise BS, Lopez MK, et al. Development and evalua-
tion of an enzyme-linked immunosorbent assay for the serodiagno-
sis of pythiosis in dogs. J Vet Intern Med. 2002;16:142-146.
20. Hummel J, Grooters A, Davidson G, et al. Successful management
of gastrointestinal pythiosis in a dog using itraconazole, terbin-
afine, and mefenoxam. Med Mycol. 2011;49:539-542.
21. Hubert JD, Grooters AM. Treatment of equine pythiosis. Compend
Contin Ed Pract Vet. 2002;13:187-194.
22. Wanachiwanawin W, Mendoza L, Visuthisakchai S, et  al. Effi-
cacy of immunotherapy using antigens of Pythium insidio-
sum in the treatment of vascular pythiosis in humans. Vaccine.
2004;22:3613-3621.
23. Grooters AM, Hodgin EC, Bauer RW, et  al. Clinicopathologic
findings associated with Lagenidium sp. infection in six dogs:
initial description of an emerging oomycosis. J Vet Intern Med.
2003;17:637-646.
24. Hillier A, Kunkle GA, Ginn PE, et al. Canine subcutaneous zygo-
mycosis caused by Conidiobolus sp.: a case report and review
of Conidiobolus infections in other species. Vet Dermatol.
1994;5:205-213.
25. Hawkins EC, Grooters AM, Cowgill ES, et  al. Treatment of
Conidiobolus sp. pneumonia with itraconazole in a dog receiv-
ing immunosuppressive therapy. J Vet Intern Med. 2006;20:
1479-1482.
26. Greene CE, Brockus CW, Currin MP, et al. Infection with Basid-
iobolus ranarum in two dogs. J Am Vet Med Assoc. 2002;221:
528-532:500.
27. Miller RI, Turnwald GH. Disseminated basidiobolomycosis in a
dog. Vet Pathol. 1984;21:117-119.
28. Cunha SC, Aguero C, Damico CB, et  al. Duodenal perfora-
tion caused by Rhizomucor species in a cat. J Feline Med Surg.
2011;13:205-207.
29. Nielsen C, Sutton DA, Matise I, et  al. Isolation of Cokeromyces
recurvatus, initially misidentified as Coccidioides immitis, from
peritoneal fluid in a cat with jejunal perforation. J Vet Diagn Invest.
2005;17:372-378.
30. Wray JD, Sparkes AH, Johnson EM. Infection of the subcutis of the
nose in a cat caused by Mucor species: successful treatment using
posaconazole. J Feline Med Surg. 2008;10:523-527.
31. Miller RI, Campbell RS. The comparative pathology of equine
cutaneous phycomycosis. Vet Pathol. 1984;21:325-332.
CHAPTER 70

Protothecosis
Jane E. Sykes

may not inactivate Prototheca.2 Prototheca spp. also colonize

Overview of Protothecosis the skin and gastrointestinal and respiratory tracts of humans.3
First Described: Prototheca spp. was first isolated from tree The algae are round to oval, 5 to 30 µm in diameter, have
slime in the 1880s. Naturally occurring protothecosis a thick cell wall, and reproduce asexually by endosporulation
was not described until 1952, in cattle (mastitis). Canine (internal division), which results in a sporangium that con-
disease was reported in 1969, 5 years after the first tains up to multiple sporangiospores. Rupture of the sporan-
description of human protothecosis (Van Kruiningen and gium releases sporangia into tissues, and the cycle continues.
othes).1 Although the classification of Prototheca species is still in flux,
six species are currently recognized: Prototheca zopfii, Proto-
Cause: Prototheca spp. (lower algae, Chlorophyceae)
theca wickerhamii, Prototheca blaschkeae, Prototheca stag-
Affected Hosts: Dogs, cats, cattle, humans, other domestic nora, Prototheca ulmea, and Prototheca cutis (Table 70-1).4
and wild animal species Only P. zopfii and P. wickerhamii have been identified as
Geographic Distribution: Worldwide except Antarctica, but causes of disease in dogs and cats. Two genotypes of P. zopfii
especially in warm, humid climates and where there is are recognized. P. zopfii genotype 1 is a weakly pathogenic
organic matter with a high water content variant and has been isolated from a minority of protothecal
mastitis cases in dairy cows.4,5 In contrast, P. zopfii genotype 2
Mode of Transmission: Cutaneous inoculation of organisms may be the most virulent Prototheca variant. It causes the vast
into tissues, or possibly systemic invasion in the face majority of bovine Prototheca mastitis cases worldwide and
of immunosuppression by organisms that have been most protothecosis in dogs.4 P. wickerhamii is often associ-
ingested or colonize the intestinal tract ated with cutaneous disease and has to date been isolated from
Major Clinical Signs: Cutaneous nodules or masses or sys- all affected cats, some dogs, and almost all human patients
temic illness with weight loss, blindness due to ocular (see Table 70-1). In dogs, disease caused by P. wickerhamii
involvement, polyuria and polydipsia, hematochezia, may follow a less aggressive clinical course than that caused by
vomiting, lameness, neurologic signs P. zopfii genotype 2.6
Differential Diagnoses: Chlorellosis (a rare algal disease), a Protothecosis is an uncommon and sporadic disease of dogs
variety of fungal diseases (e.g., histoplasmosis, cryptococ- and is rare in cats. In dogs, protothecosis is usually a serious
cosis, sporotrichosis), intestinal pythiosis, neoplasia (espe-
cially round cell neoplasia), toxoplasmosis, neosporosis,
granulomatous colitis of boxer dogs, inflammatory bowel TABLE 70-1
disease
Human Health Significance: Human infections occur as
Major Species (and Genotypes) of Prototheca and
a result of exposure to organisms in the environment Their Clinical Relevance
and are often associated with underlying immune
compromise. Host Species
Species Infected Clinical Manifestations
P. zopfii Cattle Mastitis (low
­genotype 1 ­pathogenicity)
Etiology and Epidemiology P. zopfii Dogs, humans, Mastitis (cattle),
­genotype 2 cattle, other ­disseminated infections
Protothecosis is an uncommon cutaneous or systemic disease (dogs)
caused by Prototheca species, which are unicellular algae. Pro- P. wickerhamii Dogs, cats Cutaneous disease
totheca spp. lack chlorophyll, and as a result they are dependent
P. blaschkeae Cattle, humans Mastitis (cattle), ­cutaneous
on a saprophytic lifestyle. Although ubiquitous in nature, they
disease (humans)
are especially prevalent in warm, humid climates (e.g., southern
and southeastern United States, northeastern Australia, south- P. stagnora Nonpathogenic NA
ern continental Europe, Japan) in aqueous environments where P. ulmea Nonpathogenic NA
decaying organic matter is present. They have been isolated from P. cutis Humans Cutaneous disease
tree slime, sewage, fresh and saltwater sources, fish tanks, soil,
animal excrement, and foodstuffs. Routine water chlorination NA, Not applicable.

679
680 SECTION 3  Fungal and Algal Diseases
disseminated disease, but localized cutaneous disease occurs
occasionally. Most affected dogs do not have a history of
immunosuppressive drug therapy or illness. Boxer dogs and col-
lies may be predisposed,6 possibly secondary to an underlying
genetic immunodeficiency, although a variety of other small-
and large-breed dogs also can be affected. Occasionally proto-
thecosis occurs in dogs in conjunction with infection by other
opportunistic pathogens such as Neospora,7 which supports the
likelihood that underlying immune deficiency is present. At the
author’s hospital, disseminated protothecosis was diagnosed in
a canine renal transplant recipient that was treated with cyclo-
sporine, and lymphocutaneous P. wickerhamii infection was
diagnosed in a dog treated with chemotherapy for lymphoma.7
The median age of affected dogs in one series of 17 dogs was 4
years (range, 18 months to 11 years), and approximately 70%
of the affected dogs were female.6 Cats with protothecosis have
single or multiple nodular skin lesions; disseminated disease has
not been reported. Affected cats are typically FIV and FeLV
negative, are otherwise in good health, and have ranged in age
from 3 to 16 years.8-13
Clinical Features
Signs and Their Pathogenesis
The pathogenesis of protothecosis is not fully understood.
Infection may occur secondary to cutaneous inoculation of
organisms, which may be followed by development of local-
ized cutaneous nodules or dissemination through lymphatic or
hematogenous spread. It is also possible that systemic invasion
by organisms that are ingested or colonize the gastrointestinal
tract occurs. An incubation period of 10 days to several weeks
has been suggested for human protothecosis, based on recollec-
tion of penetrating injuries before onset of disease.2
In dogs, Prototheca can disseminate to a variety of tissues,
but especially the colon, eyes, brain and meninges, kidneys,
and long bones. This results in clinical signs of inappetence and
weight loss; acute or chronic large bowel diarrhea (often with
hematochezia and tenesmus); blindness; neurologic signs such
as obtundation, seizures, or ataxia; polyuria and polydipsia;
and sometimes lameness due to osteomyelitis.6,13-16 Deafness is
often reported in association with disseminated protothecosis
and may occur as a result of central nervous system (CNS) or
inner ear involvement.13,16,17 Ocular lesions include granuloma-
tous chorioretinitis, uveitis, and panophthalmitis. Other affected
organs include the liver, skeletal muscle, thyroid gland, lymph
nodes, spleen, pancreas, stomach, small intestine, omentum,
myocardium, aorta, spinal cord, and/or lungs.16-19 Involvement
of these organs may lead to other signs such as vomiting, small
bowel diarrhea, and/or melena. Sudden death has also been
reported, possibly secondary to myocardial involvement.6,20,21
Physical Examination Findings
Cutaneous lesions in dogs are nodular and may ulcerate.
They often involve the footpads or distal limbs and associated
draining lymph nodes, but involvement of other sites such as
the trunk and planum nasale can also occur. In cats, single or
multiple firm and sometimes ulcerated cutaneous nodules may
be present. Lesions may be located on the head, footpads, and
distal limbs and tailbase8-10 and can resemble those caused by
Cryptococcus or Sporothrix.
Dogs with disseminated disease may be obtunded and/or dehy-
drated and can have a thin body condition. Some dogs are febrile,
but rectal temperature may also be normal. Ocular involvement
may be manifested by blindness or evidence of conjunctivitis,
hyphema, uveitis, cataract formation, panophthalmitis, or granu-
lomatous chorioretinitis, often with retinal detachment. Arrhyth-
mias with pulse deficits may be present in dogs with protothecal
myocarditis.7 Neurologic signs include obtundation, blindness,
deafness, ataxia, abnormal placing reactions, circling, head tilt,
nystagmus, strabismus, and abnormalities of cranial nerve func-
tion such as absent or decreased facial sensation, palpebral, men-
ace, or gag reflexes.6,14,22-25 Dried fecal material or blood may
be present around the anus, and rectal examination may reveal
the presence of hematochezia, increased fecal mucus, or melena.
Diagnosis
Laboratory Abnormalities
Complete Blood Count and Serum Biochemical Tests
The CBC in dogs with disseminated protothecosis is often nor-
mal. Occasionally it reveals nonregenerative anemia and/or
leukocytosis due to a neutrophilia and sometimes monocyto-
sis.15,16,18 Mild bandemia and/or eosinophilia may be present in
some dogs. The serum biochemistry panel may also be normal
or reveal hyperglobulinemia due to a polyclonal gammopathy.
Mild to moderate azotemia may be evident in dogs with renal
involvement.15,18 Hypoalbuminemia and hypocholesterolemia
may occur with severe bowel involvement.6
Urinalysis
The urinalysis in dogs with disseminated protothecosis may be
unremarkable or reveal isosthenuria, hematuria, pyuria, pro-
teinuria, and the presence of Prototheca organisms in the sedi-
ment.15 Urinalysis and urine culture should be performed in all
dogs suspected to have protothecosis, because more than half of
affected dogs shed algae into the urine.
Cerebrospinal Fluid Cytology
CSF analysis in dogs with CNS protothecosis may be normal or
reveal markedly increased total nucleated cell count (sometimes
>1000 cells/µL) and increased CSF protein concentration (often
>500 mg/dL).18,22,23 Eosinophilic pleocytosis has been described
in some dogs,22,25 but eosinophils are not always present.23 Pro-
totheca algae may also be found in the CSF.22,23
Diagnostic Imaging
Plain Radiography
Thoracic radiographs in dogs with disseminated prototheco-
sis are usually unremarkable.15,18,19,21 Radiographs of affected
long bones may reveal periosteal proliferative and/or osteolytic
lesions due to osteomyelitis.6
Sonographic Findings
Findings on abdominal ultrasound examination in dogs with dis-
seminated protothecosis include hyperechogenicity of the renal
cortices,15 a thickened colonic wall with loss of normal bowel
wall layering,6 and abdominal lymphadenopathy. Some dogs
have no abdominal sonographic abnormalities despite the pres-
ence of disseminated disease with colonic and renal involvement.7
Other Imaging
Colonoscopy in dogs with protothecosis that has disseminated
to the large intestine may reveal a hyperemic, friable, thickened,
irregular, and ulcerated mucosa.6
 CHAPTER 70  Protothecosis 681

Magnetic resonance imaging of the brain of one dog with
CNS involvement revealed mild to moderate ventriculomegaly,
mild protrusion of the cerebellar vermis through the foramen
magnum, syringomyelia, meningeal contrast enhancement, and
a focus of increased signal intensity in the brain on T2-weighted
and fluid attenuated inversion recovery (FLAIR) sequences.23
As it enlarged, the focus developed contrast enhancement and
caused deformation of a lateral ventricle.
Microbiologic Tests
Specific diagnostic assays for protothecosis are described in
Table 70-2.
Cytologic Examination
Prototheca organisms may be seen using cytologic examina-
tion of rectal scrapings; fine-needle aspirates of lymph nodes
or bone or skin lesions; CSF or vitreal or aqueous humor;
and urine sediment. The organisms are spheroid, ovoid, bean-
shaped or elliptical, and range in diameter from 1.5 to 30 µm
(P. zopfii) or up to 10 µm (P. wickerhamii). They have a thick,
hyaline cell wall that does not take up stain, and a basophilic,
granular cytoplasm. Multiple daughter cells (2 to usually
<10) may be seen inside the cell (Figure 70-1). In the case of
P. wickerhamii, the endospores are arranged symmetrically like
a daisy or soccer ball, whereas the endospores of P. zopfii are

TABLE 70-2
Diagnostic Assays Available for Protothecosis
Assay Specimen Type Target Performance
Cytologic exami- Aspirates of affected tissues, Prototheca algae Usually large numbers of organisms are present, so sensi-
nation urine sediment, vitreous or tivity is high, but false negatives can still occur (such as
aqueous humor, CSF when urine sediment or CSF are examined). Organisms
may be confused with other fungi or Chlorella species,
so confirmation of the diagnosis requires culture.
Culture Urine, blood, CSF, vitreous or Prototheca Prototheca spp. grow rapidly (usually within 3 days) on
aqueous humor, aspirates or ­species routine media such as blood agar and Sabouraud dex-
biopsies of affected tissues trose agar. Allows species identification and subsequent
genotyping.
Histopathology Skin or colonic biopsies, Prototheca algae Usually large numbers of organisms are present, so
enucleated eyes, tissues col- sensitivity is high. Organisms are highlighted with PAS
lected at necropsy or silver stains. Organisms may be confused with other
fungi or Chlorella species, so confirmation of protothe-
cosis requires culture.

A B
FIGURE 70-1  A, Cytology of a subretinal aspirate from an 8-year-old intact male mixed breed dog with panophthalmitis due to Prototheca spp. infection. Moderate numbers of oval
to bean-shaped organisms are present in a proteinaceous background that contained variably degenerate and ruptured neutrophils. The organisms have a thin clear capsule with deep
purple cytoplasmic granulation. Sometimes endosporulation is evident (arrows). Wright’s stain, 1000× oil magnification. B, Cytology of a bone aspirate from a 2-year-old female mixed
breed dog that developed acute vision loss. Physical examination revealed bilateral retinal detachment and a nonpainful swelling of the right distal medial humerus. Abundant numbers of
Prototheca algae are present. (Courtesy Dr. Emily Pieczarka.)
682 SECTION 3  Fungal and Algal Diseases

distributed randomly.2 The algae may be mistaken for fungi
such as Blastomyces, Coccidioides, or Cryptococcus. Because
large numbers of organisms are typically present, the sensitiv-
ity of cytologic examination for diagnosis of algal infection
is high. However, failure to find organisms does not rule out
protothecosis.
Culture
Prototheca species can be readily cultured in the microbiol-
ogy laboratory on standard media (such as blood agar and
Sabouraud dextrose agar), and yeast-like colonies are usually
visible within 7 days. Suitable specimens for culture include
aspirates or biopsies of affected tissue and body fluids such as
urine, CSF, and vitreous or aqueous humor. Prototheca zopfii
was also cultured from the blood of a dog with disseminated
disease.21 Biochemical testing of isolates allows identification
of the Prototheca species present and excludes other possible
causes such as the green alga Chlorella, a very rare cause of dis-
seminated disease in dogs that can closely resemble Prototheca
in its microscopic appearance.2,26 Differentiation of P. zopfii
genotypes requires PCR and ribosomal RNA gene sequenc-
ing. Matrix-assisted laser desorption/ionization–time of flight
(MALDI-TOF) mass spectrometry offers promise for rapid
identification to the species and genotype level in the clinical
microbiology laboratory in the future.4,27
Antifungal susceptibility testing has been performed for Pro-
totheca but has not been standardized, and the clinical relevance
of the results is not clear.2
Molecular Diagnosis Using the Polymerase Chain Reaction
Real-time PCR assays have been developed that detect and dif-
ferentiate Prototheca species,28 but these are not available on a
commercial basis for diagnosis of protothecosis in animals.
Pathologic Findings
Gross pathologic findings in dogs with disseminated protothe-
cosis include enlarged lymph nodes and gray, tan, or white nod-
ular lesions or streaks in affected organs.13,16,18,25 The colonic
wall may be thickened and contain bloody fluid. Malacic foci
may be present in the brain, or the brain may appear normal
grossly, even when meningoencephalitis is present.7,24 Histo-
pathologic examination of affected tissues typically reveals
large numbers of sporulating and nonsporulating algae accom-
panied by a mixed inflammatory response and foci of necro-
sis (Figure 70-2, A). In some tissues the inflammatory response
can be minimal to absent.16,18 Organisms may be seen free and

A B

C
FIGURE 70-2  Histopathology of the renal lymph node of a 6-year-old spayed female shih tzu with protothecosis that was being treated with cyclosporine after renal transplanta-
tion. Numerous Prototheca organisms are present. The organisms range in diameter from 5 to 15 µm and have several morphologic forms. Sporulating forms are up to 15 µm in diameter
and contain two to four wedge-shaped endospores (daughter cells). Many of the organisms present as clear, refractile, oval to polyhedral bodies composed of a thick wall and devoid of
cytoplasm and nuclei. A, H&E stain. B, Periodic acid–Schiff stain. C, Gomori’s methenamine silver.

within macrophages. The cell wall stains well with Gomori’s
methenamine silver and periodic acid–Schiff (PAS) stain, but in
contrast to Chlorella, cytoplasmic granules of Prototheca are
PAS negative. Cutaneous lesions in cats have been associated
with a granulomatous inflammatory response with multinucle-
ated giant cells and, in some cases, necrosis and neutrophilic
inflammation.9,11
Treatment and Prognosis
Surgical excision can be curative for localized cutaneous
protothecosis and should be performed whenever possible.9
Medical treatment of protothecosis is challenging. In human
patients, treatment is generally with amphotericin B, which
in some cases results in complete cure. Addition of a tetra-
cycline has also been recommended.2 Outcomes with azole
antifungals alone are often poor, although clinical responses
occur in some dogs with itraconazole alone.7 In one human
patient, a combination of amikacin and tetracycline led to
cure.29 Treatment of canine disseminated protothecosis with
amphotericin B leads to remission in some cases, but relapse
typically occurs when the drug is discontinued, or dogs fail
to respond to treatment and ultimately die or are eutha-
nized as a result of the disease.6,21 One dog with protothe-
cal meningoencephalitis appeared to improve clinically after
treatment with oral itraconazole and intrathecal amphoteri-
cin B, but was euthanized 4 weeks later because of progres-
sive disease.23
CASE EXAMPLE
Signalment: “Merlot,” a 3-year-old female spayed Siberian
husky dog from San Francisco, CA
History: Merlot was evaluated at the University of California,
Davis, Veterinary Medical Teaching Hospital for a 1-month
history of hemorrhagic diarrhea, inappetence, and
weight loss. Initially the diarrhea contained fresh blood
and mucus but more recently contained only blood. The
diarrhea occurred frequently and in small quantities.
The owner reported that Merlot had lost approximately
5 kg over the past month and had increased thirst and
urination. There had been no history of vomiting, and
Merlot’s activity level was normal. Her diet consisted of a
commercial dry dog food.
At a local veterinary clinic, multiple fecal flotations had been
negative, and Merlot had been treated with metronidazole,
aminopentamide, bismuth subsalicylate, enrofloxacin, lop-
eramide, tetracycline, metoclopramide, and a highly digest-
ible diet without clinical response. A barium series had re-
cently been performed, and findings were consistent with
gastroenteritis.
Merlot had moved to San Francisco from Massachusetts
4 months before the clinical signs began. On the way over
the owner stopped in Texas, where Merlot became lost in
a field for 30 minutes. The owner reported that Merlot was
taken to a dog park in San Francisco several times a week. No
other history of illness existed.
CHAPTER 70  Protothecosis 683
Other treatments that may be required for protothecosis
include intravenous fluid therapy, antiemetics, topical predniso-
lone acetate ophthalmic drops for uveitis, or ocular enucleation
to control pain and/or remove a focus of infection that fails to
respond to chemotherapy (see Case Example). Other treatments
such as metronidazole, sulfasalazine, or dietary manipulation
may help to control signs of protothecal colitis.
Public Health Aspects
Protothecosis is an uncommon disease of humans and most
often caused by P. wickerhamii. Infection is acquired from envi-
ronmental sources, and direct transmission of other animals
or humans does not appear to be important. Mortality is low
(<5%), and two thirds of infections are chronic and localized
to the skin.2 Joint infections and tendonitis have been described
after surgical or accidental wounds; some have been traced back
to cleaning an aquarium.30 Disseminated infections and a syn-
drome called olecranon bursitis (which follows elbow trauma)
account for the remaining third of cases. Underlying local or
systemic immune defects can be identified in many patients;
topical, local, or systemic glucocorticoid treatment is the most
frequently identified risk factor. Other immunosuppressive dis-
orders that may predispose humans to protothecosis include
diabetes mellitus and malignancy. Severe disseminated disease
has been described in bone marrow and kidney transplant recip-
ients,31,32 in those with long-standing indwelling catheters or
endotracheal tubes, and rarely in AIDS patients.30,33
Physical Examination:
Body Weight: 18.2 kg.
General: Bright, alert, responsive, and hydrated. T = 102.3°F
(39.1°C), HR = 80 beats/min, panting, mucous membranes
pink, CRT = 1 s.
Integument, Eyes, Ears, Nose, and Throat: Dried barium was
present on the dog’s lips, and there were no other clinically
significant findings. Fundoscopic examination revealed a
region of retinal separation in the left ocular fundus that
was adjacent to, dorsomedial to, and approximately the size
of the optic disc. The lesion appeared to contain yellowish
fluid.
Musculoskeletal: Thin but well-muscled. Body condition score
was 3/9.
Cardiorespiratory: No clinically significant findings.
Gastrointestinal/Genitourinary: No masses were palpated in
the abdomen. The dog vocalized and appeared painful on
caudal abdominal palpation. Frank blood was present on
the glove after rectal examination.
Peripheral Lymph Nodes: All peripheral lymph nodes were
normal in size.
Laboratory Findings:
CBC:
HCT 48.6% (40%-55%)
MCV 70.9 fL (65-75 fL)
MCHC 34.6 g/dL (33-36 g/dL)
WBC 12,100 cells/µL (6000-13,000 cells/µL)
Neutrophils 9438 cells/µL (3000-10,500 cells/µL)
Lymphocytes 2057 cells/µL (1000-4000 cells/µL)
684 SECTION 3  Fungal and Algal Diseases
Monocytes 363 cells/µL (150-1200 cells/µL)
Eosinophils 242 cells/µL (0-1500 cells/µL)
Platelets 259,000/µL (150,000-400,000
platelets/µL).
Serum Chemistry Profile:
Sodium 150 mmol/L (145-154 mmol/L)
Potassium 4.3 mmol/L (3.6-5.3 mmol/L)
Chloride 114 mmol/L (108-118 mmol/L)
Bicarbonate 28 mmol/L (16-26 mmol/L)
Phosphorus 4.2 mg/dL (3.0-6.2 mg/dL)
Calcium 9.8 mg/dL (9.7-11.5 mg/dl)
BUN 21 mg/dL (5-21 mg/dL)
Creatinine 1.2 mg/dL (0.3-1.2 mg/dL)
Glucose 99 mg/dL (64-123 mg/dL)
Total protein 6.4 g/dL (5.4-7.6 g/dL)
Albumin 2.9 g/dL (3.0-4.4 g/dL)
Globulin 3.5 g/dL (1.8-3.9 g/dL)
ALT 61 U/L (19-67 U/L)
AST 42 U/L (19-42 U/L)
ALP 126 U/L (21-170 U/L)
Cholesterol 145 mg/dL (135-361 mg/dL)
Total bilirubin 0 mg/dL (0-0.2 mg/dL).
Urinalysis: SGr 1.005; pH 6.0; no protein (SSA), bilirubin,
hemoprotein, or glucose; 0-1 WBC/HPF, rare RBC/HPF, rare
transitional epithelial cells; no crystals, casts or bacteria.
Moderate numbers of encapsulated organisms were present
that measured 10 × 17 µm.
Imaging Findings: Abdominal ultrasound examination: No
abnormalities were identified.
Microbiologic Testing: Centrifugal zinc sulfate fecal
flotation: Negative, but yeast-like bodies were observed.
Giardia and Cryptosporidium fluorescent antibody testing (fecal
specimen): Negative.
Cytologic examination of rectal scrapings: Moderate numbers
of neutrophils, small numbers of gram-positive coccobacilli,
and small numbers of what appeared to be algae were pres-
ent.
Aerobic bacterial urine culture: Small numbers of Prototheca
zopfii.
Algal susceptibility testing: MICs for amphotericin B, flucon-
azole, and itraconazole were 0.5 µg/mL, >64 µg/mL, and 1
µg/mL, respectively.
Histopathology (rectal biopsy obtained by proctoscopy): ero-
sive and hemorrhagic colitis, eosinophilic and lymphoplas-
macytic, moderate, with marked expansion of the lamina
propria by intralesional spherical organisms (protothecal
colitis).
Aerobic bacterial and fungal culture of a rectal biopsy: Small
numbers of mixed enteric species and P. zopfii.
Diagnosis: Disseminated P. zopfii infection with ocular and
colonic involvement.
Treatment: Merlot was initially treated with lipid-complexed
amphotericin B (3 mg/kg IV) on a Monday, Wednesday,
and Friday basis for five doses, after which increased
serum creatinine concentration was noted (1.7 mg/dL).
She was also treated with oxytetracycline (21 mg/kg PO
q8h). She was discharged after 11 days with instructions
to administer itraconazole (5 mg/kg PO q12h) and
lufenuron (5 mg/kg PO q24h). At a recheck examination
2 weeks later, the owners reported Merlot’s appetite had
improved slightly and that she was defecating once to
twice a day. Each bowel movement was followed by a
small amount of diarrhea at the end of defecation, with
bloody mucoid material occasionally present. In the
2 weeks since her previous visit, a foxtail had been removed
from her right pelvic limb and she had been sprayed in
the face by a skunk. Physical examination revealed a body
weight of 19.2 kg (1 kg weight gain), but aqueous flare and
multifocal retinal separations were present in both eyes.
Urinalysis was unremarkable, and rectal scrapings showed no
evidence of algal organisms. A biochemistry panel showed
normalization of the creatinine and mildly increased activity
of ALT, likely secondary to itraconazole administration. Topical
prednisolone acetate ophthalmic drops and two additional
amphotericin B treatments were administered, but bilateral
retinal detachment, blindness, then bilateral hyphema and
apparent ocular pain developed despite this treatment.
Bilateral enucleation was performed. Histopathology of the
globes showed bilateral granulomatous uveitis and retinitis
with intralesional Prototheca organisms. In the left eye,
posterior synechiae, intraocular hemorrhage, and cataract
formation were identified.
At a recheck examination 3 months after enucleation, Merlot
was eating well and had no signs of large bowel diarrhea.
Her body weight was stable and physical examination, a
CBC, a biochemistry panel, and cytologic examination of a
rectal scraping were unremarkable. Itraconazole treatment
was continued at a lower dose (5 mg/kg PO q24h), and the
lufenuron was discontinued. Two months later, and shortly
after a new puppy was introduced, Merlot developed vomit-
ing and diarrhea with small amounts of hematochezia, and
her body weight dropped to 17.5 kg. A serum biochemistry
panel showed mild hypoalbuminemia (2.6 g/dL). Cytologic
examination of a rectal scraping again revealed Prototheca
organisms, and a fecal flotation was negative. Merlot was
treated with fenbendazole and the dose of itraconazole was
increased to 7.5 mg/kg PO daily, after which diarrhea resolved.
Four months later (14 months after initial diagnosis), the dog
remained in remission but was subsequently lost to follow-up.
Comments: Diagnosis of protothecosis in this dog was
straightforward and initially based on routine urine sediment
examination. Culture was then used to identify the causative
agent as P. zopfii. Colonic involvement was confirmed
using proctoscopy and biopsy. The most likely place that
Prototheca infection was acquired was in Texas, but it is
possible that infection was acquired elsewhere. Partial
clinical remission was observed after treatment with lipid-
complexed amphotericin B and itraconazole, but enucleation
was required to control pain. Remission was prolonged (>1
year) even though only seven amphotericin B treatments
were administered in the first 2 months after diagnosis.
Lufenuron is a chitin synthetase inhibitor. Chitin is present in
the cell walls of some algae (including Chlorella), but whether
Prototheca possess chitin in their cell walls is not clear.

SUGGESTED READINGS
Lass-Florl C, Mayr A. Human protothecosis. Clin Microbiol Rev.
2007;20:230-242.
Quigley RR, Knowles KE, Johnson GC. Disseminated chlorellosis in a
dog. Vet Pathol. 2009;46:439-443.
Stenner VJ, Mackay B, King T, et al. Protothecosis in 17 Australian dogs
and a review of the canine literature. Med Mycol. 2007;45:249-266.
REFERENCES
1. Van Kruiningen HJ, Garner FM, Schiefer B. Protothecosis in a dog.
Pathol Vet. 1969;6:348-354.
2. Lass-Florl C, Mayr A. Human protothecosis. Clin Microbiol Rev.
2007;20:230-242.
3. Sonck CE, Koch Y. Prototheca as parasites of skin. Mykosen.
1971;14:475-482.
4. Ahrholdt J, Murugaiyan J, Straubinger RK, et al. Epidemiological
analysis of worldwide bovine, canine and human clinical Proto-
theca isolates by PCR genotyping and MALDI-TOF mass spec-
trometry proteomic phenotyping. Med Mycol. 2012;50:234-243.
5. Ricchi M, Goretti M, Branda E, et al. Molecular characterization
of Prototheca strains isolated from Italian dairy herds. J Dairy Sci.
2010;93:4625-4631.
6. Stenner VJ, Mackay B, King T, et  al. Protothecosis in 17 Aus-
tralian dogs and a review of the canine literature. Med Mycol.
2007;45:249-266.
7. Sykes JE. Unpublished observations, 2012.
8. Coloe PJ, Allison JF. Protothecosis in a cat. J Am Vet Med Assoc.
1982;180:78-79.
9. Dillberger JE, Homer B, Daubert D, et al. Protothecosis in two cats.
J Am Vet Med Assoc. 1988;192:1557-1559.
10. Endo S, Sekiguchi M, Kishimoto Y, et  al. The first case of feline
Prototheca wickerhamii infection in Japan. J Vet Med Sci.
2010;72:1351-1353.
11. Finnie JW, Coloe PJ. Cutaneous protothecosis in a cat. Aust Vet J.
1981;57:307-308.
12. Kaplan W, Chandler FW, Holzinger EA, et al. Protothecosis in a
cat: first recorded case. Sabouraudia. 1976;14:281-286.
13. Migaki G, Font RL, Sauer RM, et al. Canine protothecosis: review
of the literature and report of an additional case. J Am Vet Med
Assoc. 1982;181:794-797.
14. Lane LV, Meinkoth JH, Brunker J, et  al. Disseminated protothe-
cosis diagnosed by evaluation of CSF in a dog. Vet Clin Pathol.
2012;41:147-152.
15. Pressler BM, Gookin JL, Sykes JE, et al. Urinary tract manifesta-
tions of protothecosis in dogs. J Vet Intern Med. 2005;19:115-119.
16. Gaunt SD, McGrath RK, Cox HU. Disseminated protothecosis in a
dog. J Am Vet Med Assoc. 1984;185:906-907.
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17. Cook Jr JR, Tyler DE, Coulter DB, et al. Disseminated protothe-
cosis causing acute blindness and deafness in a dog. J Am Vet Med
Assoc. 1984;184:1266-1272.
18. Rakich PM, Latimer KS. Altered immune function in a dog with dis-
seminated protothecosis. J Am Vet Med Assoc. 1984;185:681-683.
19. Buyukmihci N, Rubin LF, DePaoli A. Protothecosis with ocular
involvement in a dog. J Am Vet Med Assoc. 1975;167:158-161.
20. Blogg JR, Sykes JE. Sudden blindness associated with protothecosis
in a dog. Aust Vet J. 1995;72:147-149.
21. Moore FM, Schmidt GM, Desai D, et  al. Unsuccessful treatment
of disseminated protothecosis in a dog. J Am Vet Med Assoc.
1985;186:705-708.
22. Gupta A, Gumber S, Bauer RW, et  al. What is your diagnosis?
Cerebrospinal fluid from a dog. Eosinophilic pleocytosis due to
protothecosis. Vet Clin Pathol. 2011;40:105-106.
23. Young M, Bush W, Sanchez M, et al. Serial MRI and CSF analysis
in a dog treated with intrathecal amphotericin B for protothecosis.
J Am Anim Hosp Assoc. 2012;48:125-131.
24. Salvadori C, Gandini G, Ballarini A, et  al. Protothecal granu-
lomatous meningoencephalitis in a dog. J Small Anim Pract.
2008;49:531-535.
25. Tyler DE, Lorenz MD, Blue JL, et al. Disseminated protothecosis
with central nervous system involvement in a dog. J Am Vet Med
Assoc. 1980;176:987-993.
26. Quigley RR, Knowles KE, Johnson GC. Disseminated chlorellosis
in a dog. Vet Pathol. 2009;46:439-443.
27. von Bergen M, Eidner A, Schmidt F, et al. Identification of harm-
less and pathogenic algae of the genus Prototheca by MALDI-MS.
Proteomics Clin Appl. 2009;3:774-784.
28. Ricchi M, Cammi G, Garbarino CA, et al. A rapid real-time PCR/
DNA resolution melting method to identify Prototheca species.
J Appl Microbiol. 2011;110:27-34.
29. Zhao J, Liu W, Lv G, et al. Protothecosis successfully treated with
amikacin combined with tetracyclines. Mycoses. 2004;47:156-158.
30. Pascual JS, Balos LL, Baer AN. Disseminated Prototheca wick-
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2004;31:1861-1865.
31. Lass-Florl C, Fille M, Gunsilius E, et  al. Disseminated infection
with Prototheca zopfii after unrelated stem cell transplantation for
leukemia. J Clin Microbiol. 2004;42:4907-4908.
32. Mohd Tap R, Sabaratnam P, Salleh MA, et  al. Characterization
of Prototheca wickerhamii isolated from disseminated algaemia
of kidney transplant patient from Malaysia. Mycopathologia.
2012;173:173-178.
33. Kaminski ZC, Kapila R, Sharer LR, et  al. Meningitis due to
Prototheca wickerhamii in a patient with AIDS. Clin Infect Dis.
1992;15:704-706.
CHAPTER 71
Pneumocystosis
Remo Lobetti
Overview of Pneumocystosis
First Described: Pneumocystis was first discovered in Brazil
in 1909 (Chagas), when it was initially mistaken for a try-
panosome.1 It was first reported in dogs in the 1950s.2
Causes: Pneumocystis spp. (an ascomycete)
Affected Hosts: Dogs, humans, pigs, horses, goats, nonhu-
man primates; Pneumocystis species appear to have host
specificity.
Geographic Distribution: Worldwide
Mode of Transmission: Opportunistic invasion of immuno-
suppressed hosts after transmission by aerosol
Major Clinical Signs: Weight loss, respiratory distress, exercise
intolerance, variable cough
Differential Diagnoses: Canine distemper virus infection,
­bacterial pneumonia, mycobacterial infections, other
fungal causes of pneumonia, pulmonary neoplasia (espe-
cially lymphoma), parasitic lung disease, acute respiratory
­distress syndrome, pulmonary fibrosis
Human Health Significance: Severely immunocompro-
mised humans are at risk for pneumocystosis, but only
P. ­jirovecii infects humans. Whether humans can become
colonized with Pneumocystis strains that infect dogs is
not known.
Etiologic Agent and Epidemiology
Pneumocystis is a saprophyte of low virulence with a world-
wide distribution. Its primary habitat is the mammalian lung,
where it causes opportunistic pneumonia. Pneumocystis pneu-
monia has been reported in dogs, pigs, horses, goats, primates,
and humans. Subclinical or latent infections are common in
rats, mice, guinea pigs, rabbits, cats, sheep, and various wildlife
species. Most reports of pneumonia due to Pneumocystis spp.
infection are linked with documented or suspected immunodefi-
ciency syndromes in the host.3
The taxonomy of Pneumocystis is uncertain. Previously it
was classified as a unicellular protozoan in the phylum Sar-
comastigophora. However, ultrastructurally the reproductive
behavior of Pneumocystis is similar to the ascospore formation
by yeasts, and its organelles and staining properties by light
microscopy resemble those of pathogenic fungi. Phylogenetic
classification based on 16S-like rRNA sequences indicates that
Pneumocystis is most closely related to the fungi of the class
Ascomycetes, especially Saccharomyces cerevisiae, and so it has
been reclassified as fungus, “albeit an odd one.”4 Biologically,
686
it behaves similarly to a protozoan, because it is susceptible to
drugs used to treat protozoal infections but resistant to most
antifungal drugs. The morphology of Pneumocystis organisms
and the histopathology of the lesions they produce in humans
and animals throughout the world are similar. Although con-
troversial, five species of Pneumocystis have been described,
which appear to be highly host specific: two species that infect
rats, P
­ neumocystis carinii and Pneumocystis wakefieldiae; one
species that infects mice, Pneumocystis murina; a species that
infects rabbits, Pneumocystis oryctolagi; and P ­ neumocystis jir-
ovecii (pronounced “yee row vet zee”), which infects humans.5-7
Species designations for the organisms isolated from dogs
and other animals require further genetic and biologic study,
although DNA analysis of Pneumocystis that infected a Cava-
lier King Charles spaniel suggested that dogs are infected with a
unique species, with the proposed name of Pneumocystis canis.8
Biologic differences between isolates from different hosts are
supported by the relative difficulty of experimental cross-host
species transmission. Because historically P. carinii was thought
to cause Pneumocystis pneumonia in humans, Pneumocystis
pneumonia has long been referred to as “PCP.” Despite the
name change to P. jirovecii in the 1970s, the term PCP has
been retained for convenience. For purposes of this chapter, the
organism will be referred to simply as Pneumocystis. Strain dif-
ferences may also exist within species of Pneumocystis.
Pneumocystis appears to be maintained in nature by trans-
mission from infected to susceptible hosts. The primary mode of
spread is thought to be airborne droplet transmission.9 Sporadic
disease may occur as a result of activation of latent infection by
stress, crowding, and immunosuppressive therapy during hospi-
talization. Clinical disease can also be experimentally activated
by treatment with glucocorticoids or cytotoxic chemotherapy. A
higher prevalence of infection was found in dogs with distemper
compared with a corresponding control population.10 Genetic
analysis of isolates from humans suggests that recent acquisition
of infection from another person, rather than reactivation of
infection, may be responsible for clinical illness.11,12
Most affected dogs have been young miniature dachshunds
(less than 1 year of age) or Cavalier King Charles spaniels.8,13-17
Affected miniature dachshunds and Cavalier King Charles span-
iels may possess an underlying primary immune deficiency.17,18
Clinical Features
Signs and Their Pathogenesis
Pneumocystis is inhaled and can colonize the lower respiratory
tracts of clinically healthy mammals; however, it rarely multiplies
to large numbers in the lungs of healthy hosts. Transmission of
infection to neonates can occur via aspiration of contaminated
amniotic fluid when the placenta is infected. When impaired

host resistance (especially reduced CD4 ­T-lymphocyte counts)
or preexisting pulmonary disease is present, rapid proliferation
of organisms can occur.19 The entire life cycle of Pneumocystis is
completed within alveolar spaces (Figure 71-1). In this location,
two main forms, the trophozoite (1 to 4 µm in diameter) and
cyst (8 µm), are found. Trophozoites adhere closely to alveo-
lar epithelial cells and are thought to undergo sexual reproduc-
tion by conjugation, with formation of a diploid zygote. The
zygote becomes a sporocyte (or precyst) and undergoes meio-
sis followed by mitosis. This leads to formation of a mature,
thick-walled cyst or spore case, which contains eight haploid
ascospores. The cyst ruptures, and the spores transform into tro-
phozoites, which replicate asexually to form more trophozoites,
or sexually to form more cyst forms. The transmissible form has
not been identified.7
The overgrowth and clustering of Pneumocystis within
alveolar spaces leads to blockage of capillaries and decreased
gas exchange. Intra-alveolar organisms are often accompanied
by thickening of alveolar septa, but they seldom invade the pul-
monary interstitium and are rarely phagocytosed by alveolar
Mitochondrion
Nucleus
A Asexual Haploid
trophic form
Conjugation
Excystment
Diploid
precyst
Meiosis
Early cyst
Maturation
B Sexual reproduction
FIGURE 71-1  Life cycle of Pneumocystis species. Trophozoites adhere to alveolar epi-
thelial cells in the lungs and undergo both asexual (A) and sexual (B) reproduction. Sexual
reproduction is initiated by conjugation, which results in formation of a zygote. Meiosis
occurs followed by mitosis, which leads to the formation of 8 ascospores within a mature
cyst. The cyst ruptures, releasing spores that become trophozoites. (Modified from Walzer
PD, Smulian AG. Pneumocystis species. In: Mandell GL, Bennett JE, Dolin R, eds. Principles
and Practice of Infectious Diseases, 7 ed. Philadelphia, PA: Elsevier; 2010:3377-3390.)
CHAPTER 71  Pneumocystosis 687
macrophages. With an adequate immune response, the body
eliminates the infection, but the removal of large numbers of
organisms and cellular debris may take up to 8 weeks. Both the
presence of the organisms and the mild inflammatory response
they provoke contribute to pulmonary damage.
Although Pneumocystis infections are usually confined to
the lungs, organisms are rarely reported in extrapulmonary
sites. In humans, extrapulmonary pneumocystosis occurs pri-
marily when there is overwhelming pulmonary infection, pro-
found underlying immunodeficiency, and after long-term use
of aerosolized pentamidine for prophylaxis against P. jirovecii
pneumonia in HIV-infected patients.20 Sites of extrapulmonary
infection include lymph nodes, spleen, liver, bone marrow, gas-
trointestinal tract, eyes, thyroid gland, adrenal glands, kidneys,
heart, pancreas, and external auditory canal. Extrapulmonary
pneumocystosis has been described in one dog.
The typical clinical history in dogs with pneumocystosis is
that of gradual weight loss and respiratory difficulty that pro-
gresses over 1 to 4 weeks. The weight loss, which occurs in spite
of a good appetite in most dogs, may be associated with diar-
rhea and occasional vomiting. Cough is not always reported,
but reduced exercise tolerance is uniform. Infected animals
often show a minimal or temporary response to antibiotic and
­glucocorticoid therapy.
Physical Examination Findings
Abnormalities on physical examination of dogs with pneu-
mocystosis include dyspnea, tachycardia, and increased dry
respiratory sounds on thoracic auscultation. Animals are often
in poor condition and cachectic and can show dermatologic
changes, such as superficial bacterial pyoderma and demodico-
sis (Figure 71-2). Although the mucous membranes are gener-
ally of normal color, they may be cyanotic in severely affected
animals. Affected dogs remain relatively alert and afebrile,
although mild pyrexia may be present.
FIGURE 71-2  One-year-old dachshund with pneumocystosis. Note the thin body
condition and alopecia secondary to generalized demodectic mange.
688 SECTION 3  Fungal and Algal Diseases
FIGURE 71-3  Lateral thoracic radiographs from a 1-year-old male neutered Cavalier
King Charles spaniel with pneumocystosis. A diffuse marked interstitial pattern is present.
Mild right-sided cardiomegaly was also identified.
Diagnosis
Laboratory Abnormalities
Complete Blood Count
Hematologic abnormalities in dogs with pneumocystosis are
nonspecific. Neutrophilic leukocytosis is the most consistent
finding. Less frequently, eosinophilia or monocytosis are found.
Lymphocyte counts can be elevated, normal, or low. Polycythe-
mia may occur secondary to arterial hypoxemia from impaired
gaseous exchange. Thrombocytosis is often present in miniature
dachshunds. Artifactual thrombocytopenia and megathrombo-
cytosis have been observed in Cavalier King Charles spaniels,
which may have a hereditary basis that is likely unrelated to the
suspected immunodeficiency.
Serum Chemistry Profile
Total serum proteins are usually within reference limits in dogs
with pneumocystosis, with a low to borderline-low globulin
level, which correlates with low γ-globulin levels on serum pro-
tein electrophoresis.17 In some dogs, mild hyperglobulinemia is
present.21 Mild hypoalbuminemia may also be detected. Arte-
rial hypoxemia, hypocapnia, and increased arterial blood pH
indicate an uncompensated respiratory alkalosis. The arterial
pO2 is often lower than expected based on the clinical signs and
thoracic radiographs.
Diagnostic Imaging
Findings on thoracic radiography include diffuse, bilaterally
symmetric, miliary-interstitial to alveolar lung disease, with
compensatory emphysema in severely infected animals.22 Inter-
stitial patterns are most common (Figure 71-3). Solitary lesions,
unilateral involvement, cavitary lesions, spontaneous pneumo-
thorax, and lobar infiltrates have also been described. Tracheal
elevation, right-sided heart enlargement, and pulmonary arte-
rial enlargement reflect cor pulmonale secondary to diffuse pul-
monary disease. Computed tomography (CT) scans may show
diffuse pulmonary interstitial infiltrates and small focal regions
of increased opacity (Figure 71-4), but CT findings in dogs with
pneumocystosis have not been extensively described.
FIGURE 71-4  Thoracic CT scan from a 4-year-old female spayed Cavalier King Charles
spaniel with pneumocystosis that was evaluated for lethargy, inappetence, and respira-
tory distress. There is a diffuse interstitial pattern and focal peripheral alveolar infiltrates
(arrowheads).
Microbiologic Tests
Diagnostic assays available for pneumocystosis in dogs or cats
are described in Table 71-1. Identification of pneumonia in a
Cavalier King Charles spaniel or miniature dachshund should
always raise suspicion for pneumocystosis.
Cytologic Diagnosis
The diagnosis of pneumocystosis requires direct demonstration
of Pneumocystis cysts in biopsy specimens, respiratory fluids, or
occasional extrapulmonary sites. Transtracheal, endotracheal,
or bronchoalveolar lavage specimens, lung aspirates, and oro-
pharyngeal secretions may contain organisms. Transtracheal
lavage specimens have been useful for detection of organisms
in dogs.17 No cytologic technique is as reliable or as definitive
as histopathologic examination of lung biopsy specimens for
documentation of active Pneumocystis infection. However, lung
biopsy is more invasive, has potential complications of hemor-
rhage or pneumothorax, and is associated with additional costs
and hospitalization. Pneumocystis organisms are difficult to
detect in respiratory secretions or lavage specimens, and success
in finding them usually depends on the experience of the exam-
iner and the collection and processing of specimens. Polychrome
stains, such as Wright’s, Giemsa, and methylene blue, will dem-
onstrate nuclei of trophozoites and intracystic sporozoites in
cytologic specimens, but the walls of cysts and trophozoites will
not be apparent (Figure 71-5). Diff Quik stain may be useful to
rapidly identify organisms.23
Serologic Diagnosis
Serologic assays are available for detection of human infections
with P. jirovecii; however, their diagnostic value is uncertain,
since many immunodeficient humans who develop pneu-
mocystosis fail to produce antibody titers, and healthy indi-
viduals frequently have high titers. Increased antibody titers
to P
­ neumocystis persist for long periods, offering a valuable
 CHAPTER 71  Pneumocystosis 689

TABLE 71-1
Diagnostic Assays Available for Pneumocystosis in Dogs or Cats
Assay Specimen Type Target Performance
Cytologic examination Lung aspirates; transtracheal, Pneumocystis False negatives can occur when specimen size is small or
endotracheal, or bronchoal- trophozoites low numbers of organisms are present. Identification to
veolar lavage specimens and cysts the genus level requires immunostaining or PCR assay.
PCR assays Lung aspirates; transtracheal, Pneumocystis The sensitivity and specificity of assays may vary de-
endotracheal, or bronchoal- spp. DNA pending on assay design. Assays designed to detect
veolar lavage specimens P. jirovecii may not detect organisms that infect dogs.
In the absence of cytologic evidence of Pneumocystis
infection, the significance of a positive PCR result
must be interpreted in light of clinical findings, be-
cause of the potential for subclinical colonization.
Histopathology Lung biopsy or necropsy Pneumocystis Large numbers of organisms are usually found filling
specimens trophozoites alveolar spaces. Cyst forms are best demonstrated
and cysts with silver stains or periodic acid–Schiff. The identity
of the organisms can be confirmed with immunohisto-
chemistry or PCR assay.

Molecular Diagnosis Using the Polymerase Chain Reaction

PCR assays have been used to detect Pneumocystis in bron-
choalveolar lavage specimens from humans12,27-29 and in lung
tissue from dogs.16,30 The sensitivity of PCR assays on bron-
choalveolar lavage fluids from infected humans is greater than
that of conventional staining.31 DNA analysis of PCR products
has been used to type isolates from humans and animals.8 Some
PCR assays (especially specific real-time PCR assays) that detect
P. jirovecii may not detect Pneumocystis organisms that infect
dogs because of sequence differences.

FIGURE 71-5  Impression smear of lung tissue from a 1-year-old male neutered Cava-
lier King Charles spaniel with pneumocystosis. Large foamy and activated macrophages
are present, as well as fewer neutrophils and lymphocytes. Abundant trophozoites are
present and range in size from 1 to 2 µm (arrowhead). Organisms can also be seen within
a cyst (arrow). Wright’s stain.
index of infection in epidemiologic studies. However, they
are of limited use for an immediate diagnosis.24 Circulating
Pneumocystis antigen has been detected in human serum by
counter-immunoelectrophoresis and ELISA methods. How-
ever, antigenemia also is found in up to 15% of clinically
healthy humans. Direct fluorescent antibody assays can specifi-
cally detect organisms in sputum, tracheal aspirates, or pulmo-
nary tissue.25 Immunoperoxidase techniques also can be used
to identify Pneumocystis in impression smears or formalin-
fixed, paraffin-embedded lung sections.26
Fungal Culture
Pneumocystis cannot be cultured in the laboratory from clinical
specimens.
Immunologic Findings
The majority of the immunologic studies of pneumocystosis
in dogs have been done in the miniature dachshund.17,32 Lym-
phocyte stimulation assay results, using phytohemagglutinin
and pokeweed mitogen, show severe immunosuppression,
especially when compared to controls, even though a normal
lymphocyte count can be present. Immunoglobulin fraction
quantification consistently reveals deficiencies of IgA, IgM, and
IgG. Low globulin levels, immunoglobulin deficiencies, and
decreased lymphocyte function have been reported in the Cava-
lier King Charles spaniel.18,30 The absence of immunoglobulins
in the presence of pathologic changes is a significant finding,
since affected dogs have chronic infections, which should
result in an immunoglobulin response. Immunoglobulin defi-
ciencies are still present after the Pneumocystis and skin infec-
tions have resolved, which supports the presence of a primary
defect. Lymphocyte transformation studies and the results of
immunoglobulin quantification suggest that both T- and B-cell
abnormalities exist in affected dogs. Serum complement activ-
ity in affected miniature dachshunds has been normal. CD3
and CD79a lymphocyte staining of lymph nodes and spleen
shows marked absence of B cells with the presence of T cells.
Immunologic abnormalities in the miniature dachshund are
similar to those described in human patients that have common
variable immunodeficiency syndrome (also known as acquired
or adult-onset hypogammaglobulinemia). Common variable
immunodeficiency syndrome is a primary immunodeficiency
690 SECTION 3  Fungal and Algal Diseases
disease characterized by little or no antibody production by B
lymphocytes, normal or decreased numbers of B lymphocytes,
and abnormal T-lymphocyte function.33
Pathologic Findings
Gross Pathologic Findings
Pathologic findings in pneumocystosis are primarily confined
to the lungs, although dissemination to regional lymph nodes,
spleen, liver, bone marrow, and other organs has been reported.
On gross examination, the lungs are firm, consolidated, and pale
brown or gray. They do not collapse when the chest cavity is
opened. Unlike many pneumonic processes, fluid is not expressed
from cut surfaces of the lung. The pulmonary and mediastinal
lymph nodes are often enlarged. Small amounts of fluid may be
found in the pleural cavity. Cardiac enlargement, when present,
has been right sided in all cases.
Histopathologic Findings
On histologic examination, alveolar spaces are filled with
aggregates of amorphous, foamy, eosinophilic material that
has a frothy, honeycomb-like pattern (Figure 71-6). A few
macrophages and detached alveolar lining cells may be pres-
ent, but polymorphonuclear leukocytes are absent. Special
stains are needed to identify cyst forms. Little or no phagocy-
tosis of intact Pneumocystis organisms is present. However,
nonviable organisms (such as those seen after treatment) are
often phagocytosed, and macrophages may contain Gomori’s
methenamine silver (GMS)-positive granular material that
represents the residuum of cyst wall degradation. In some
instances, alveolar septa are markedly thickened by dense
accumulations of plasma cells, lymphocytes, and macrophages
(lymphoplasmacytic pneumonia). The septa may be widened
by fibrosis in chronic infections. With GMS stain, cyst forms
are spherical, ovoid, or crescent-shaped structures that range
from 4 to 7 µm in diameter and have dot-like, argyrophilic
(stain with silver), focal cyst wall thickenings. Cyst walls also
can be demonstrated with other stains such as toluidine blue,
and they fluoresce when stained with orange G of a Papani-
colaou stain. The internal structure of trophozoites in tissue
sections and smears are best demonstrated with Giemsa stain.
On ultrastructural examination, intact alveoli are filled with
compact aggregates of trophozoite and cyst forms. Trophozoites
commonly line alveoli.
Appropriate histologic staining is essential to ensure detec-
tion of Pneumocystis organisms. Routine hematoxylin and
eosin stain does not readily demonstrate the developmental
forms of Pneumocystis. Only the nuclei of intracystic sporo-
zoites and trophozoites are stained. Various modifications of
methenamine silver staining can be employed to stain the cyst
walls brownish-black; trophozoites will not be detected. The
cyst wall also stains with periodic acid–Schiff stain (see Figure
71-6).
Treatment and Prognosis
Specific antimicrobial chemotherapy is most beneficial when
pneumocystosis is suspected (usually based on the affected dog
breed) or diagnosed during its early stages. The treatment of
choice is trimethoprim and a sulfonamide (TMS). Pentamidine
isethionate (an aromatic diamidine) has also been effective in
humans, but TMS is more effective and less toxic. TMS has also
been used as prophylaxis in immunodeficient humans.
FIGURE 71-6  Histopathology of the lung of an 18-month-old male neutered
Cavalier King Charles spaniel with pneumocystosis. Foamy, honeycomb-like material
fills the alveoli. Periodic acid–Schiff stain.
Treatment with TMS has been reported in 8 dogs;16,17,22 4
were dachshunds that subsequently recovered.22 A TMS dose
of 15 mg/kg, every 8 hours, or 30 mg/kg, every 12 hours, for 3
weeks was used; the longest reported follow-up in these studies
was for 4 months. Folic acid supplementation should be given
if adverse effects such as leukopenia and anemia are observed
or if long-term therapy is required. In human infections, resis-
tance to sulfonamides has been noted among some strains of
P. jirovecii.34
Other treatment options include atovaquone, clindamy-
cin and primaquine, and dapsone-trimethoprim combinations.
Atovaquone is licensed for the treatment of human pneumo-
cystosis.35 This drug, which also has been used to treat canine
babesiosis (see Chapter 10), is not as effective against Pneu-
mocystis infections in humans as pentamidine or TMS but has
lower toxicity. In humans, pentamidine or atovaquone has also
been administered by aerosol, but this is less effective than oral
administration and is not recommended.36,37
Combination therapy with clindamycin and primaquine is
now the preferred alternative to treatment with TMS in humans
(e.g., for patients intolerant of TMS)37 but has not been reported
for treatment of dogs. Dapsone and trimethoprim or pyrimeth-
amine, in combination, have been effective in experimental ani-
mals and in clinical trials in immunosuppressed humans with
pneumocystosis.38 Pneumocystis is resistant to azole antifungal
drugs and amphotericin B (because it lacks ergosterol in its cell
wall), but the anthelmintics benzimidazole and albendazole
have been effective in experimental infections.39,40
Nonspecific immunostimulants such as cimetidine and
levamisole have been given adjunctively to treat affected min-
iature dachshunds,41 without much success. Supportive care is
essential for any patient with Pneumocystis pneumonia because
of disturbed alveolar gaseous exchange. Supplemental oxygen
therapy may be needed, and ventilatory assistance may also be
required. Immunosuppressive agents should be discontinued
if possible. Antimicrobial chemotherapy of pulmonary pneu-
mocystosis results in a decline in arterial oxygen related to the
inflammatory reaction to dying organisms. Administration of
anti-inflammatory doses of glucocorticoids during specific treat-
ment for P. jirovecii infection improves pulmonary function and
survival in humans.37,42-44

Public Health Aspects
Pneumocystis organisms are ubiquitous in the environment.
However, molecular and epidemiologic evidence suggests that
spread may occur between humans because of localized out-
breaks. Humans and animals are exposed to the same envi-
ronmental sources of Pneumocystis. Pneumocystis species that
infect dogs are likely different from those that infect humans,
CASE EXAMPLE
Signalment: “Tessa”, a 1-year-old intact female miniature
dachshund (see Figure 71-2)
History: Tessa was evaluated for a history of chronic tachypnea
and weight loss. The clinical signs had been nonresponsive
to various treatments that had included multiple
antibiotics, bronchodilators, mucolytics, multivitamins, and
glucocorticoids. From 2 months of age, she had a history of
recurrent infections including otitis externa, hemorrhagic
enteritis, tonsillitis, folliculitis, and pyogranulomatous
dermatitis. These conditions had responded each time to
supportive care.
Physical Examination:
Body Weight: 4.5 kg.
General: Quiet, alert, and responsive. Ambulatory on all four
limbs. Rectal temperature = 101.8°F (38.8°C), HR = 138
beats/min, RR = 129 breaths/min, mucous membranes pink,
CRT = 1 s.
Integument: A poor coat was present with focal alopecia on
the head and back.
Musculoskeletal: Body condition score was 3/9 with diffuse
and symmetrical muscle atrophy.
Respiratory: An increased respiratory rate with increased
abdominal effort was noted; tracheal palpation elicited a
dry cough.
All Other Systems: No clinically significant abnormalities were
noted.
Laboratory Findings:
CBC:
HCT 54% (40-55%)
MCV 68 fL (65-75 fL)
MCHC 35.7 g/dL (33-36 g/dL)
WBC 34,600 cells/µL (6000-15,000 cells/µL)
Neutrophils 23,200 cells/µL (3000-11,000 cells/µL)
Band neutrophils 700 cells/µL (0-300 cells/µL)
Lymphocytes 5500 cells/µL (1000-4800 cells/µL)
Monocytes 4800 cells/µL (100-2000 cells/µL)
Basophils 350 cells/µL (100 cells/µL)
Platelets 730,000 platelets/µL (200,000-500,000 platelets/µL).
SUGGESTED READINGS
Chabe M, Aliouat-Denis CM, Delhaes L, et  al. Pneumocystis: from a
doubtful unique entity to a group of highly diversified fungal species.
FEMS Yeast Res. 2011;11:2-17.
Watson PJ, Wotton P, Eastwood J, et al. Immunoglobulin deficiency in
Cavalier King Charles Spaniels with Pneumocystis pneumonia. J Vet
Intern Med. 2006;20:523-527.
CHAPTER 71  Pneumocystosis 691
but further studies are required to verify this. The primary
risk factor for clinical pneumocystosis in humans is immuno-
deficiency (especially HIV infection, hematologic malignancies
and solid tumors, transplant recipients, and collagen vascular
disorders).37 Thus, the greatest risk for acquisition of infec-
tion from a pet would be if the animal was clinically ill from
Pneumocystis pneumonia and the person in close contact was
immunocompromised.
Serum Chemistry Profile:
Sodium 147 mmol/L (145-154 mmol/L)
Potassium 4.6 mmol/L (3.6-5.3 mmol/L)
Phosphorus 4.8 mg/dL (3.0-6.2 mg/dL)
Calcium 10.9 mg/dL (9.7-11.5 mg/dL)
BUN 17 mg/dL (5-21 mg/dL)
Creatinine 0.5 mg/dL (0.3-1.2 mg/dL)
Glucose 132 mg/dL (64-123 mg/dL)
Total protein 5.1 g/dL (5.3-7.5 g/dL)
Albumin 3.5 g/dL (2.3-3.5 g/dL)
Globulin 1.6 g/dL (2-3.7 g/dL)
ALT 65 U/L (10-60 U/L)
ALP 210 U/L (100-250 U/L)
Cholesterol 196 mg/dL (135-361 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.2 mg/dL)
α-globulins 0.8 g/dL (0.8-1.6 g/dL)
β-globulins 0.6 g/dL (0.7-1.3 g/dL)
γ-globulins 0.3 g/dL (0.4-1.1 g/dL).
Urinalysis: SGr 1.045; pH 6.0, negative for protein, bilirubin,
blood, and glucose. Inactive sediment.
Imaging Findings: On thoracic radiographs the
cardiovascular structures appeared within normal limits.
A diffuse interstitial pulmonary pattern was present.
Cytology Findings: Transtracheal wash cytology demon-
strated active, blastic alveolar macrophages with phagocyt-
ic vacuolization, large numbers of neutrophils, and many
free and phagocytosed Pneumocystis organisms.
Aerobic Bacterial Culture: Culture of the transtracheal
wash fluid yielded no growth after 48 hours’ incubation.
Fecal Examination: Fecal flotation: Negative for parasites.
Diagnosis: Pneumocystis spp. pneumonia.
Treatment: The dog was treated with a potentiated
sulfonamide at 15 mg/kg PO q8h for 3 weeks, which resulted
in a complete recovery.
Comments: This dog was diagnosed with pneumocystosis as
a result of a primary immune deficiency disease, most likely
common variable immunodeficiency syndrome. The latter
would also explain the history of chronic skin infections and
gastroenteritis. CD3 and CD79a lymphocyte staining of a
peripheral lymph node biopsy showed a marked absence of
B cells.
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16. Sukura A, Saari S, Jarvinen AK, et al. Pneumocystis carinii pneumonia
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18. Watson PJ, Wotton P, Eastwood J, et  al. Immunoglobulin defi-
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19. Kelly MN, Shellito JE. Current understanding of Pneumocystis
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21. Sykes JE. Unpublished observations, 2012.
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23. Cregan P, Yamamoto A, Lum A, et al. Comparison of four meth-
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25. Ng VL, Virani NA, Chaisson RE, et al. Rapid detection of Pneumo-
cystis carinii using a direct fluorescent monoclonal antibody stain.
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26. Kondo H, Hikita M, Ito M, et al. Immunohistochemical study of Pneu-
mocystis carinii infection in pigs: evaluation of Pneumocystis pneu-
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27. Kitada K, Oka S, Kimura S, et al. Detection of Pneumocystis cari-
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28. Lu JJ, Chen CH, Bartlett MS, et  al. Comparison of six different
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29. Roux P, Lavrard I, Poirot JL, et al. Usefulness of PCR for detection
of Pneumocystis carinii DNA. J Clin Microbiol. 1994;32:2324-2326.
30. Hagiwara Y, Fujiwara S, Takai H, et  al. Pneumocystis carinii
pneumonia in a Cavalier King Charles Spaniel. J Vet Med Sci.
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31. Flori P, Bellete B, Durand F, et al. Comparison between real-time
PCR, conventional PCR and different staining techniques for diag-
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lavage specimens. J Med Microbiol. 2004;53:603-607.
32. Lobetti R. Common variable immunodeficiency in miniature
dachshunds affected with Pneumocystis carinii pneumonia. J Vet
Diagn Invest. 2000;12:39-45.
33. Cunningham-Rundles C, Knight AK. Common variable immune
deficiency: reviews, continued puzzles, and a new registry. Immu-
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34. Calderon E, de la Horra C, Montes-Cano MA, et  al. [Genotypic
resistance to sulfamide drugs among patients with Pneumocystis
jiroveci pneumonia]. Med Clin (Barc). 2004;122:617-619.
35. Stoeckle M, Tennenberg A. Atovaquone for Pneumocystis carinii
pneumonia. Ann Intern Med. 1995;122:314:author reply 314–315.
36. Chan C, Montaner J, Lefebvre EA, et al. Atovaquone suspension
compared with aerosolized pentamidine for prevention of Pneu-
mocystis carinii pneumonia in human immunodeficiency virus–
infected subjects intolerant of trimethoprim or sulfonamides.
J Infect Dis. 1999;180:369-376.
37. Walzer PD, Smulian AG. Pneumocystis species. In: Mandell GL,
Bennett JE, Dolin R, eds. Principles and Practice of Infectious Dis-
eases. 7th ed. Philadelphia, PA: Elsevier; 2010:3377-3390.
38. Hughes WT. Use of dapsone in the prevention and treatment
of Pneumocystis carinii pneumonia: a review. Clin Infect Dis.
1998;27:191-204.
39. Bartlett MS, Queener SF, Shaw MM, et al. Pneumocystis carinii is
resistant to imidazole antifungal agents. Antimicrob Agents Che-
mother. 1994;38:1859-1861.
40. Bartlett MS, Edlind TD, Lee CH, et al. Albendazole inhibits Pneu-
mocystis carinii proliferation in inoculated immunosuppressed mice.
Antimicrob Agents Chemother. 1994;38:1834-1837.
41. Farrow BR, Watson AD, Hartley WJ, et al. Pneumocystis pneumo-
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42. Consensus statement on the use of corticosteroids as adjunctive
therapy for Pneumocystis pneumonia in the acquired immu-
nodeficiency syndrome. The National Institutes of Health–
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Adjunctive Therapy for Pneumocystis Pneumonia. N Engl J Med.
1990;323:1500-1504.
43. Bozzette SA, Finkelstein DM, Spector SA, et al. A randomized trial
of three anti-Pneumocystis agents in patients with advanced human
immunodeficiency virus infection. NIAID AIDS Clinical Trials
Group N Engl J Med. 1995;332:693-699.
44. Masur H. Prevention and treatment of Pneumocystis pneumonia.
N Engl J Med. 1992;327:1853-1860.

Protozoal Diseases
CHAPTER 72
Toxoplasmosis
Michael R. Lappin
Overview of Toxoplasmosis
First Described: The organism that was ultimately named
Toxoplasma gondii was first described in France in 1908
(Nicolle and Manceaux)1
Cause: Toxoplasma gondii, a coccidial protozoan parasite
(phylum Apicomplexa)
Affected Host Species: Cats are the definitive host and are
the only species known to complete the sexual phase of
T. gondii culminating in the passage of oocysts in feces.
Cats and most other vertebrates can serve as intermedi-
ate hosts; invertebrates can serve as transport hosts by
mechanical carriage of T. gondii oocysts.
Geographic Distribution: T. gondii has a worldwide distribu-
tion except in the absence of cats
Major Clinical Signs: Fever, ocular inflammation, ataxia, sei-
zures, muscle pain, and respiratory distress are the most
common clinical signs in cats; dogs have similar signs but
develop illness less frequently than cats.
Differential Diagnoses: In the dog, Neospora caninum induces
the most similar clinical signs; many other chronic intra-
cellular bacterial infections and fungal infections in dogs
and cats need to be considered a differential diagno-
ses for many of the clinical and laboratory abnormities
induced by T. gondii.
Human Health Significance: There is significant risk to the
fetus after transplacental infection and to any immuno-
compromised person.
Etiologic Agent and Epidemiology
Toxoplasma gondii is a coccidian that is one of the most prev-
alent parasites infecting warm-blooded vertebrates around
the world.2-4 Only cats complete the sexual phase in the
SECTION 4
gastrointestinal tract (also known as the enteroepithelial cycle)
and pass environmentally resistant oocysts in feces (Figure
72-1). Sporozoites develop within shed oocysts after 1 to 5 days
of exposure to oxygen and appropriate environmental tempera-
ture and humidity (Figure 72-2). This process is known as sporu-
lation. After oocyst ingestion, released sporozoites can penetrate
the intestinal tracts of cats or intermediate hosts and disseminate
in blood or lymph as tachyzoites during active infection. T. gon-
dii can penetrate most mammalian cells and replicates asexually
within infected cells until the cell is destroyed. If an appropriate
immune response occurs, replication of tachyzoites is attenuated
and slowly dividing bradyzoites develop that persist within cysts
in extraintestinal tissues. These tissue cysts form readily in the
central nervous system (CNS), muscles, and visceral organs. Live
bradyzoites may persist in tissue cysts for the life of the host.
It is likely that most T. gondii–infected cats and dogs harbor
tissue cysts for life. Thus, the presence of serum antibodies is
likely to indicate current infection. T. gondii seroprevalence rates
vary by the lifestyle of the cat or dog. In general, increased sero-
prevalence correlates with increased age as a result of increased
risk of exposure over time. It also correlates with outdoor expo-
sure, since animals with access to the outdoors are most likely
to contact infected intermediate hosts. In a recent study of clini-
cally ill cats, T. gondii antibodies were detected in 31.6% of the
12,628 cats tested.5 The seroprevalence was lowest in regions
with low humidity (Figure 72-3). In another study of feral cats in
Florida, T. gondii antibodies were detected in 12.1% of the cats.6
Although T. gondii infection is common among cats, the oocyst
shedding period is generally less than 21 days. Thus, detection of
T. gondii oocysts in feline feces is uncommon. For example, in
two studies in the United States, T. gondii oocysts were detected
in feces of fewer than 1% of cats.7,8
Dogs do not produce T. gondii oocysts, but they can
mechanically transmit oocysts after they ingest feline feces.9
The tissue phases of T. gondii infection occur in dogs and may
be associated with clinical disease. Approximately 20% of dogs
in the United States are seropositive for T. gondii antibodies.10
Before 1988, many dogs diagnosed with toxoplasmosis based
693
694 SECTION 4  Protozoal Diseases

FIGURE 72-1  Life cycle of Toxoplasma gondii. Cats are most often infected when they ingest bradyzoite cysts in tissues of prey. The bradyzoites transform into merozoites and undergo
repeated cycles of schizogony in the cat’s intestinal tract. The merozoites then transform into microgametes (male) and macrogametes (female). When a microgamete penetrates a mac-
rogamete, a zygote forms. The zygote is shed as an unsporulated oocyst in the feces, which is not infectious. Sporulation occurs after 1 to 5 days. The extraintestinal cycle occurs in cats
or other intermediate hosts after they ingest sporulated oocysts or tissue cysts. Sporozoites penetrate intestinal cells and transform into tachyzoites, which rapidly multiply in nucleated
cells throughout the body in the face of immunosuppression. This can result in severe disease in the developing fetus. Ultimately organisms are contained by the immune system within
bradyzoite cysts in muscle, brain, and visceral organs (latent infection).

15-19%
25-29%
30-34%
35-39%
40-45%
FIGURE 72-3  Continental United States regional seroprevalence in clinically ill cats.
Results were based on the presence of Toxoplasma gondii-specific IgG or IgM in serum.
Information was available from 3,640 serum specimens. (From Vollaire MR, Radecki SV,
Lappin MR. Seroprevalence of Toxoplasma gondii antibodies in clinically ill cats in the
FIGURE 72-2  Sporulated oocysts of Toxoplasma gondii. The oocysts are 10 × 12 µm United States. Am J Vet Res 2005;66:874-877.)
in diameter.
bradyzoites) or it can occur by transplacental transmission. Cats
on histologic evaluation were in fact infected with Neospora can also be infected through the transmammary route.11 In dogs,
caninum (see Chapter 73). evidence for venereal transmission also exists, and repeated
transplacental infection has been documented.12,13
Clinical Features Most cats are not coprophagic, so most are infected when
they ingest T. gondii bradyzoites in tissue cysts during carniv-
Signs and Their Pathogenesis orous feeding. After ingestion, the bradyzoites transform into
Infection of warm-blooded vertebrates occurs after ingestion of merozoites, which replicate in the epithelial cells of the intesti-
any of the three life stages of T. gondii (sporozoite, tachyzoites, nal tract, then transform into microgametes and macrogametes.

When a microgamete penetrates a macrogamete, a zygote forms.
The zygote then transforms into an unsporulated oocyst, which
is shed in feces for 3 to 21 days. After sporulation (formation
of sporozoites within the oocyst), oocysts can survive in the
environment for months to years and are resistant to most dis-
infectants. The T. gondii oocyst shedding prepatent period is
stage dependent (ingestion of bradyzoites has a shorter prepa-
tent period than ingestion of sporozoites) but is not dose depen-
dent.14 In addition, transmission of T. gondii is most efficient
when cats consume tissue cysts (carnivorism) and when interme-
diate hosts consume oocysts (fecal-oral transmission). Infection
of rodents with T. gondii leads to clinical signs of altered behav-
ior, so that the rodent becomes less fearful of cats.15 This may
increase the likelihood that the definitive host (cat) will become
infected and potentiate the sexual phase of the organism.
Whether clinical toxoplasmosis develops is dependent on both
host and parasite effects. Some strains of T. gondii may be more
pathogenic than others, and some strains may have specific tissue
affinities, such as a tendency to cause ocular disease in cats.16,17
If a poor immune response is mounted after primary infection,
overwhelming tachyzoite replication that results in tissue necrosis
is the major cause of disease.2-4 This mechanism is also likely in
cats or dogs with chronic (latent) toxoplasmosis that then become
immune suppressed. One example is activation of T. gondii
infection in cats or dogs after administration of immunosuppres-
sive drugs such as cyclosporine.18-21 Other immunosuppressive
conditions such as FIV infection can also result in activation of
toxoplasmosis.22 The mechanisms for chronic clinical toxoplas-
mosis have not been fully determined.2 T. gondii antigens and
antigen-containing immune complexes have been documented in
the serum of affected cats. Persistent infection was not associated
with chronic kidney failure in cats of one study.23-25
The large majority of cats infected with T. gondii never develop
detectable clinical abnormalities. In general, the enteroepithelial
cycle in the cat rarely leads to clinical signs. Only 10% to 20% of
experimentally inoculated cats develop self-limiting, small bowel
diarrhea for 1 to 2 weeks following primary oral inoculation with
T. gondii tissue cysts; this is presumed to be from the enteroepithe-
lial replication of the organism. T. gondii enteroepithelial stages
were found in intestinal tissues from two cats with inflammatory
bowel disease that had positive response to administration of anti-
Toxoplasma drugs.26 Eosinophilic fibrosing gastritis was recently
described in a T. gondii–infected cat.27
Fatal extraintestinal toxoplasmosis in cats can develop from
overwhelming intracellular replication of tachyzoites following
primary infection; hepatic, pulmonary, CNS, and pancreatic tissues
are commonly involved.2-4,28-30 Kittens infected by the transpla-
cental or transmammary routes develop the most severe signs
of extraintestinal toxoplasmosis and generally die of pulmonary
or hepatic disease. Common clinical signs in cats with dissemi-
nated toxoplasmosis include lethargy, anorexia, and respiratory
distress. Disseminated toxoplasmosis has been documented in
cats concurrently infected with FeLV, FIV, or feline infectious
peritonitis virus, as well as following cyclosporine administra-
tion for allergic dermatitis, immune-mediated disorders, or after
renal transplantation.18,19,31
Chronic toxoplasmosis occurs in some cats and should be
on the differential diagnosis list for cats with uveitis, chorio-
retinitis (Figure 72-4), cutaneous lesions, fever, muscle hyper-
esthesia, myocarditis with arrhythmias, weight loss, anorexia,
seizures, ataxia, icterus, diarrhea, respiratory distress, or pancre-
atitis.2-4,32-42 Toxoplasmosis appears to be a common infectious
CHAPTER 72  Toxoplasmosis 695
FIGURE 72-4  Toxoplasma gondii–associated chorioretinitis in an experimentally
inoculated cat.
cause of uveitis in cats; anterior uveitis or posterior uveitis can
occur, and the manifestations can be either unilateral or bilat-
eral.2,41,42 Kittens infected transplacentally or through the trans-
mammary route commonly develop ocular disease.16
In dogs, respiratory, gastrointestinal, or neuromuscular infec-
tion with associated signs of fever, vomiting, diarrhea, respira-
tory distress, ataxia, seizures, and icterus occurs most commonly
with generalized toxoplasmosis.2,43-50 Generalized toxoplasmo-
sis is most common in immunosuppressed dogs, such as those
with canine distemper virus infection or those treated with cyclo-
sporine to prevent renal transplant rejection. Neurologic signs
depend on the location of the primary lesions and include ataxia,
seizures, tremors, cranial nerve deficits, paresis, and paralysis.
Dogs with myositis present with weakness, stiff gait, or muscle
atrophy. Rapid progression to tetraparesis and paralysis with
lower motor neuron dysfunction can occur. One study associated
T. gondii antibodies with polyradiculoneuritis in dogs.49 Some
dogs with suspected neuromuscular toxoplasmosis probably have
neosporosis. Myocardial infection with ventricular arrhythmias
occurs in some infected dogs. Retinitis, anterior uveitis, iridocycli-
tis, nodular conjunctivitis, and optic neuritis occur in some dogs
with toxoplasmosis, but they are less common than in cats.50
Cutaneous disease also has been reported, which can manifest
as pustules, pruritus, subcutaneous nodules, and/or alopecia.21,51
Diagnosis
Diagnosis of toxoplasmosis requires interpretation of specific
microbiologic tests (Table 72-1) in light of the presence of
consistent clinical abnormalities. No clinical abnormalities are
specific for the disease.2-4
Laboratory Abnormalities
Complete Blood Count
T. gondii should be on the differential diagnosis list for cats
or dogs with appropriate clinical findings and nonregenerative
anemia, neutrophilic leukocytosis or neutropenia, lymphocyto-
sis or lymphopenia, monocytosis, and/or eosinophilia.
696 SECTION 4  Protozoal Diseases

TABLE 72-1
Microbiologic Assays for Diagnosis of Toxoplasma gondii Infection
Assay Specimen Type Assay Target Comments
Fecal flotation Feces T. gondii oocysts The negative predictive value of this assay is poor for clinical
toxoplasmosis, since most cats cease shedding by the time
illness occurs.
Fecal PCR assay Feces T. gondii DNA As for fecal flotation.
Cytologic Effusions, skin T. gondii tachyzoites Confirms current infection and is generally associated with
­examination lesions, tissue aspi- and occasionally disease. Organisms cannot be readily distinguished from
rates bradyzoites Neospora caninum.
Histopathology Multiple tissues T. gondii tachyzoites If the organisms are detected in the presence of inflammation
and bradyzoites and necrosis, clinical toxoplasmosis is likely. Can be dif-
ficult to differentiate T. gondii from tissue protozoans such
as N. caninum in dogs. Immunohistochemistry can be used
to differentiate the two species.
PCR assay Effusions, blood, T. gondii DNA T. gondii DNA can be amplified from the blood of normal
multiple tissues dogs and cats and so PCR on blood has a low positive
predictive value. Amplification of specific DNA confirms
infection, and if appropriate clinical signs and inflammation
are present, positive results document clinical toxoplasmosis.
Serology for Serum T. gondii IgM Most consistent with recent infection but can be induced dur-
T. gondii IgM antibodies ing reinfection and by other immune drugs such as glucocor-
ticoids. Positive results do not correlate with active disease.*
T. gondii IgG Serum T. gondii IgG Most consistent with infection of > 10 days duration. Positive
antibodies results do not correlate with active disease.*
Agglutination Serum T. gondii Hypothetically detects all antibody classes but can be falsely
assays antibodies negative in animals that only possess anti-Toxoplasma
IgM antibody. Positive results do not correlate with active
disease.*

*Results of serum antibody assays are combined with clinical findings to aid in making a diagnosis of clinical toxoplasmosis.

Serum Chemistry Profile Sonography

Depending on the organ system involved, cats with clinical
toxoplasmosis may have increases in serum protein and bili-
rubin concentrations, as well as increased activities of creatine
kinase, ALT, ALP, and lipase.2-4 Findings are similar for dogs;
dogs with chronic toxoplasmosis may develop hyperglobulin-
emia that is generally polyclonal.52
Urinalysis
Proteinuria and bilirubinuria have been detected in some dogs
or cats with clinical toxoplasmosis.
Cerebrospinal Fluid Analysis
CSF protein concentrations and cell counts are often higher
than normal, with the predominant white blood cells in CSF
being small mononuclear cells. However, increased neutrophils
also are commonly found in cats with acute CNS toxoplasmosis.
Increased CSF protein concentrations and mixed inflammatory
cell infiltrates occur in dogs with CNS toxoplasmosis.
Diagnostic Imaging
Radiographs
Pulmonary toxoplasmosis most commonly causes diffuse inter-
stitial to alveolar patterns (Figure 72-5); pleural effusion has
rarely been documented as well.
Ultrasound findings consistent with pancreatitis or diffuse hepa-
titis could be noted in dogs or cats with involvement of these
tissues due to T. gondii infection. Intra-abdominal lymphadeno-
megaly may be noted in dogs or cats with polysystemic disease.
Advanced Imaging
MRI findings in dogs and cats with toxoplasmosis may be unre-
markable, or focal or mass lesions may be identified in the brain
and/or spinal cord. On MRI, lesions are often isointense on
T1-weighted imaging and hyperintense on T2-weighted imag-
ing, and they may enhance peripherally or uniformly with con-
trast (Figure 72-6).53-55
Microbiologic Testing
Cytologic Diagnosis
Using cytologic examination, T. gondii bradyzoites or tachyzo-
ites can be detected in tissues, effusions, bronchoalveolar lavage
fluids, aqueous humor, or CSF (Figure 72-7).32,35,36,38 A defini-
tive antemortem diagnosis of toxoplasmosis can be made if the
organism is identified; however, this is uncommon, particularly
in association with sublethal disease. In the dog, N. caninum
tachyzoites cannot be distinguished from those of T. gondii on
cytologic examination, and so further diagnostics are needed to
make a definitive diagnosis.2
 CHAPTER 72  Toxoplasmosis 697

FIGURE 72-5  Thoracic radiographs of a 7-year-old female spayed domestic shorthair cat with pulmonic toxoplasmosis that developed one month after renal transplantation. There is
a diffuse interstitial pattern. The cat subsequently developed respiratory arrest and tachyzoites were seen in fluid that was suctioned from the endotracheal tube used for resuscitation. Nec-
ropsy revealed severe, diffuse, necrotizing pneumonia with intralesional tachyzoites. Histiocytic, lymphoplasmacytic, and necrotizing lesions with intralesional tachyzoites were also seen
in the renal allograft, ureter, bladder, liver, pancreas, peritoneum, muscularis mucosa of the stomach, and myocardium. Tachyzoites were not seen in the brain, but there was perivascular
inflammation that suggested the possibility of infection. Immunohistochemistry confirmed that the tachyzoites were T. gondii.

of oocysts in cats has a poor negative predictive value for clinical

toxoplasmosis, because most clinical toxoplasmosis results from
disseminated infections that occur after oocyst shedding has been
completed.

Serologic Diagnosis in Cats

FIGURE 72-6  T1-weighted postcontrast sagittal magnetic resonance image from an
11-year-old female spayed domestic longhair that was evaluated for a 5-day history of
inappetence, ataxia, abnormal vocalization, and circling. The cat was FIV and FeLV negative
and had no other immunosuppressive conditions. A contrast-enhancing lesion is present
in the right brainstem. CSF analysis revealed a mixed monocytic (primarily lymphocytic)
pleocytosis with a total nuclear cell count of 36 cells/µL and a total protein concentration of
78 mg/dL. Meningoencephalitis secondary to T. gondii infection was confirmed at necropsy.
Fecal Flotation
T. gondii oocysts measure 10 × 12 µm. When identified in feces
of cats with diarrhea, this suggests infection by T. gondii.2-4,56
However, Besnoitia and Hammondia infections of cats pro-
duce morphologically similar oocysts. If oocysts of this size are
detected in feces of dogs, N. caninum infection is most likely if
clinical disease is present.2 However, T. gondii oocysts can be
present if the dog has ingested infected feline feces. PCR assays
are available to differentiate T. gondii and N. caninum. Alter-
nately, serum antibody responses can be followed sequentially
to attempt to determine the infecting genus. Overall, detection
Detection of T. gondii antibodies in serum is used most fre-
quently for the diagnosis of clinical toxoplasmosis. A multitude
of different techniques have been assessed, including ELISA,
immunofluorescent antibody (IFA), Western blot immunoassay,
and a variety of agglutination tests.57-67 A latex agglutination
assay and an indirect hemagglutination assay are available com-
mercially. These assays can be applied to serum from either dogs
or cats and theoretically detect all classes of immunoglobulin
directed against T. gondii. However, they rarely detect antibody
in feline serum samples when only IgM is present.60 ELISA, IFA,
and Western blot immunoassays have been adapted to detect
IgM, IgG, and IgA antibody responses by using heavy-chain–
specific secondary antibodies.57,63,64 T. gondii–specific serum
IgA antibody responses are similar to IgG antibody responses,
and this antibody class is usually measured only in research
studies. Several commercial laboratories in the United States
offer T. gondii IgM and IgG testing by ELISA.68 The following
are common findings concerning T. gondii IgM and IgG anti-
body test results in cats.
Toxoplasma gondii IgM antibody titers. 
• Using ELISA, approximately 80% of healthy, experimen-
tally infected cats have detectable T. gondii–specific IgM in
serum within 2 to 4 weeks after inoculation with T. gon-
dii; these titers generally are negative within 16 weeks after
infection.57,58
• As occurs in some healthy women, persistent IgM titers (>16
weeks) have been documented commonly in cats co-infected
with FIV and in cats with ocular toxoplasmosis.41,65 Because
of these findings and the fact that some cats never have a
detectable IgM response, IgM titers cannot accurately be used
to predict when a cat is shedding oocysts. If a clinician is con-
cerned that an individual cat is shedding T. gondii oocysts,
fecal flotation or a fecal PCR assay should be performed.
698 SECTION 4  Protozoal Diseases
A B
FIGURE 72-7  A, Tachyzoites of Toxoplasma gondii from the peritoneal cavity of a cat with fulminant toxoplasmosis. Unstained specimen. B, Cluster of tachyzoites in a tracheobronchial
lavage specimen from a 12-year-old male neutered cat that developed respiratory distress 3 weeks after renal transplantation. Wright’s stain, 1000× oil magnification.
• In one study of cats with clinical toxoplasmosis, T. gondii
IgM titers were detected in the serum of 93% of the cats,
whereas T. gondii IgG titers were detected in only 60% of
the cats. Thus, IgM antibodies have a higher positive predic-
tive value than IgG for clinical feline toxoplasmosis.57,65
• Some cats with chronic T. gondii infections that are initially
IgM positive, but then become IgM negative, again became
IgM positive after repeat inoculation with T. gondii and
immunosuppression through inoculation with FIV and
administration of glucocorticoids.57,67 However, clinical
signs of toxoplasmosis do not develop in these cats. Thus,
presence of IgM antibodies in feline serum does not always
mean that clinical toxoplasmosis is present.
Toxoplasma gondii IgG titers.  cannot be used to make a diagnosis of feline or canine toxo-
• T. gondii–specific IgG can be detected by ELISA in serum in
the majority of healthy experimentally inoculated cats within
3 to 4 weeks after infection.57,58
• By the time IgG antibodies are detected, the oocyst shedding
period has usually been completed. Thus, IgG-seropositive
cats are of minimal public health risk.
• T. gondii IgG antibody titers can be detected for at least
6 years after infection in experimentally inoculated cats;
because the organism probably persists in tissues for life, IgG
antibodies probably do as well.62
• Single, high IgG titers do not necessarily suggest recent or
active T. gondii infection; healthy cats can have titers that
exceed 1:10,000 as long as 6 years after experimental induction
of toxoplasmosis.62
• Some cats with low T. gondii antibody titers can become
seronegative based on the cutoff value used in an individual
assay even though T. gondii is still within tissues. Based on
an approximate 10% interassay variation in the ELISA tech-
nique, some cats with low positive IgG titers (1:64) can be
positive for IgG antibodies on one analysis and negative on a
subsequent analysis or vice versa.
• An increasing T. gondii IgG titer documents recent or active
infection, but in experimentally infected cats, the time span
from the first detectable positive IgG titer to the maximal IgG
titer is approximately 2 to 3 weeks. Thus, some cats with
clinical toxoplasmosis will have reached their maximal IgG
titer by the time they are evaluated serologically.
• Rising T. gondii IgG antibody titers occur in healthy infected
cats as well as cats with clinical toxoplasmosis and so, when
assessed alone, do not prove clinical toxoplasmosis.
• In humans and cats that reactivate chronic T. gondii infection
after immune suppression, IgG titers only rarely increase.
In dogs, assays that detect IgM and IgG titers are available in some
commercial laboratories.68 There have been very few research
studies that assess canine serologic responses to T. gondii in differ-
ent situations. However, most of the findings documented in cats
appear to be true in dogs, based on the author’s clinical experience.
For the reasons just discussed, antibody test results alone
plasmosis. However, the following combination can be used to
make a presumptive antemortem diagnosis:
• Demonstration of antibodies in serum that suggest exposure
to T. gondii
• Demonstration of an IgM titer higher than 1:64, or a four-
fold or greater rise in IgG titer, which suggests recent or
active infection
• Clinical signs of disease consistent with toxoplasmosis
• Exclusion of other common causes of the clinical syndrome
• Positive response to appropriate treatment
Because T. gondii infection cannot be eliminated, most cats and
dogs will be antibody positive for life, so there is little reason
to repeat serum antibody titers after the clinical disease has
resolved or to administer drugs with T. gondii activity to cats or
dogs without clinical signs of toxoplasmosis.
Other serologic assays.  Assays that measure T. gondii anti-
gens or immune complexes have been evaluated in some cats.
However, as for antibody tests, antigen or immune complexes
can be detected in cats with or without clinical illness. These
assays are not offered commercially.23,24,57
Molecular Diagnosis Using Nucleic Acid–Based Testing
Recently, PCR assays have been used to document T. gondii DNA
in feces and can be used to differentiate T. gondii from other
organisms.56 T. gondii DNA can be amplified from the blood of

A B
FIGURE 72-8  Histopathologic findings in Toxoplasma gondii infections. A, A tissue cyst filled with Toxoplasma gondii bradyzoites in the brain tissues of an experimentally infected
mouse. B, Pancreas of the cat in Figure 72-5. There is pyogranulomatous inflammation with intralesional tachyzoites (arrows). H&E stain. C, Immunohistochemistry clearly demonstrates
the presence of T. gondii tachyzoites in the pancreas (brown staining).
TABLE 72-2
Antimicrobial Drugs That May Be Used to Treat Clinical Toxoplasmosis in Dogs and Cats
Drug Dose
Azithromycin 10 mg/kg
Clindamycin 10-12 mg/kg
Ponazuril 20 mg/kg
Trimethoprim-sulfa 15 mg/kg
If a positive response to treatment is achieved by week 4 but the animal is still improving slowly, continue treatment for 1 week past clinical resolu-
tion or when maximal response is recognized.
healthy cats and dogs, and so positive PCR assay results do not
correlate with clinical disease.69,70 Thus, PCR assays are used most
frequently with cytologic examination or histopathology to docu-
ment that the organisms seen were T. gondii, or to detect T. gondii
in aqueous humor or CSF in conjunction with serology.42,44-46,70
The combination of detection of T. gondii–specific antibody
in serum, blood, aqueous humor, or CSF and amplification of
T. gondii DNA by PCR assay is the most accurate way to diag-
nose ocular or CNS toxoplasmosis. Whereas T. gondii–specific
IgA, IgG, and DNA can be detected in aqueous humor and CSF
of both normal and clinically ill cats, T. gondii–specific IgM
has only been detected in the aqueous humor or CSF of clini-
cally ill cats and so may be the best indicator of clinical disease.
Whether this is true for dogs has not been assessed to date.
Pathologic Findings
Gross Pathologic Findings
T. gondii infection generally induces pyogranulomatous reac-
tions and necrosis. The pyogranulomas may be visualized
grossly depending on the organ involved. Effusions that are
characteristic of modified transudates or exudates may be found
on necropsy.
Histopathologic Findings
Histopathology in animals with toxoplasmosis usually reveals
pyogranulomatous inflammation with necrosis. T. gondii
tachyzoites or bradyzoites are commonly detected (Figure 72-8,
A and B). However, if necrosis is severe, the organism may be
obscured. In these cases, DNA can often be amplified from the
tissues, or the agent can be visualized after immunohistochemical
CHAPTER 72  Toxoplasmosis 699
C
Route Interval Duration
PO q24h 4 weeks
PO q12h 4 weeks
PO q24h 4 weeks
PO q12h 4 weeks
staining (Figure 72-8, C). Because T. gondii and N. caninum
induce similar syndromes in dogs, performance of PCR assays
for both organisms in dogs is indicated in order to differentiate
between them.
Treatment and Prognosis
Cats or dogs with suspected clinical toxoplasmosis should be
administered supportive care as needed. Clindamycin hydro-
chloride or a trimethoprim-sulfonamide combination has been
prescribed most frequently for the specific treatment of clinical
toxoplasmosis (Table 72-2).2-4,66 One of the drugs should be
prescribed for 1 week, since most clinical signs of toxoplas-
mosis will begin to resolve within that time period. If a positive
response is recognized, treatment should be continued for a
total of 4 weeks if possible. If there is a poor response to therapy
after the first 7 days, an alternative drug should be considered.
Recurrence of clinical signs may be more common in cats or
dogs treated for less than 4 weeks.72
Azithromycin has been used successfully in a limited num-
ber of cats, but the optimal duration of therapy is unknown.
Pyrimethamine combined with sulfa drugs is effective for the
treatment of human toxoplasmosis but commonly results in
toxicity in cats. Pyrimethamine has been administered suc-
cessfully with other drugs for the treatment of neosporosis in
dogs and so may be effective for treatment of clinical toxo-
plasmosis in this species. Ponazuril has been used experimen-
tally in T. gondii–infected rodents and should be studied for
the treatment of feline toxoplasmosis.71,73 Currently, an optimal
treatment regimen for the use of this drug for this purpose in
700 SECTION 4  Protozoal Diseases
cats is unknown. However, ponazuril (20 mg/kg PO q24h for
28 days) was apparently successful in the treatment of a dog
with suppurative keratitis and necrotizing conjunctivitis due
to T. gondii infection.50 Addition of fluconazole to pyrimeth-
amine and trimethoprim was also effective in rodent models
of toxoplasmosis and requires further study.74 Cats or dogs
with suspected Toxoplasma uveitis should be treated with anti-
Toxoplasma drugs in combination with topical or systemic glu-
cocorticoids to avoid secondary lens luxations and glaucoma.
Enucleation may be required if lens luxations and glaucoma
develop from persistent uveitis.
The prognosis is poor for cats or dogs with hepatic, CNS, or
pulmonary disease caused by tachyzoite replication, particularly
in those that are immunocompromised by anti-inflammatory
drugs or retrovirus co-infection. If they survive, cats or dogs
with CNS involvement may not experience complete resolution
of neurologic signs after treatment.
Immunity and Vaccination
There is no evidence to suggest that any drug can eliminate
T. gondii infection in dogs or cats. Thus, recurrence of disease
is always possible, and infected dogs and cats often remain sero-
positive. In cats, if repeat oocyst shedding occurs, the number of
oocysts shed is small and shedding is of short duration. Systemic
reactivation of T. gondii infection after administration of immu-
nosuppressive drugs can occur in both dogs and cats.
Several types of vaccines have been studied to block oocyst
shedding in cats, but there is no vaccine for prevention of clinical
toxoplasmosis in dogs or cats.
Prevention
To avoid exposure to T. gondii, cats and dogs should not be
allowed to hunt or be fed undercooked meats. Care should be
taken to control transport hosts such as cockroaches that have
been shown to carry T. gondii oocysts.
Public Health Aspects
Primary T. gondii infection in immunocompetent humans
results in self-limiting fever, malaise, and lymphadenopathy that
may not be recognized or is misdiagnosed. Primary infection of
mothers by T. gondii during gestation can lead to clinical toxo-
plasmosis in the fetus; stillbirth, CNS disease, and ocular disease
are common clinical manifestations (Figure 72-9). As T-helper
cell counts decline, approximately 10% of people with AIDS
develop toxoplasmic encephalitis from reactivation of bradyzo-
ites in tissue cysts.
People most commonly acquire toxoplasmosis by ingestion
of sporulated oocysts or tissue cysts, or transplacentally. To
prevent toxoplasmosis, humans should avoid consumption of
undercooked meats or ingestion of sporulated oocysts. In a
study of 6282 meat samples from 698 retail meat stores, using
a feline bioassay, T. gondii was detected in none of the beef
or chicken specimens tested and only a small number of pork
specimens.75 However, T. gondii has been detected in the tissues
of free-ranging chickens.76 Although exposure to cats is epide-
miologically associated with acquiring toxoplasmosis in some
FIGURE 72-9  Healed chorioretinitis in a 60-year-old woman who had recovered
from congenital toxoplasmosis. The infection resulted in a permanent visual deficit. (Image
courtesy Mrs. Susan Sykes.)
studies, touching individual cats is probably not a common way
to acquire toxoplasmosis for the following reasons2-4,77-81:
• Cats generally only shed oocysts for days to several weeks
after primary inoculation.
• Repeat oocyst shedding is rare, even in cats receiving glucocor-
ticoids or cyclosporine, or in those infected with FIV or FeLV.
• Cats inoculated with tissue cysts 16 months after primary
inoculation do not shed oocysts.
• Cats are very fastidious and usually do not allow feces to
remain on their skin for time periods long enough to lead to
oocyst sporulation; the organism was not isolated from the
fur of cats that had been shedding millions of oocysts 7 days
previously.
However, because some cats will repeat oocyst shedding when
exposed a second time, cat feces should always be handled care-
fully. If a fecal specimen from a cat contains oocysts that mea-
sure 10 × 12 µm, it should be assumed that the organism present
is T. gondii. The feces should be collected and removed daily
until the oocyst shedding period is complete; administration of
clindamycin (20 mg/kg PO daily) may shorten the oocyst shed-
ding period if started after infection is documented.2,82
Because humans are not commonly infected with T. gondii
through contact with individual cats, testing healthy cats for
evidence of T. gondii infection is not recommended.79 Fecal
examination is an adequate procedure to determine when cats
are actively shedding oocysts but cannot indicate when a cat has
shed oocysts in the past. There is no serologic assay that accu-
rately indicates this, and most cats that are shedding oocysts are
seronegative. Most seropositive cats have completed the oocyst
shedding period and are unlikely to repeat shedding; most sero-
negative cats would shed the organism if infected. If owners are
concerned that they may have toxoplasmosis, they should see
their physicians for testing.
Dogs do not complete the enteroepithelial phase of T. ­gondii
but can mechanically transmit oocysts after ingesting feline
feces.9

CASE EXAMPLE
Signalment: “Fluffy,” a 3-year-old male neutered Siamese mix
from Evans in northern Colorado
History: Fluffy was referred after being treated for anterior
uveitis and recurrent fever at another clinic. The clinical
signs had been noted for approximately 1 month. Fluffy
was entirely indoors in a suburban environment, but
adopted from a humane society at around 6 months of
age. His last vaccines (rabies, feline herpesvirus-1, feline
calicivirus, and panleukopenia virus) were administered
6 months before the uveitis was first noted. He was fed a
commercial processed food. There were no other animals
at the house. He was treated with selamectin monthly but
had not received his dose this month because of the fever.
The fever seemed intermittent, and when normal, Fluffy
had normal appetite and attitude. The uveitis persisted
in spite of treatment with amoxicillin-clavulanic acid
administered daily for 10 days and topical 1% prednisolone
acetate applied three times daily.
Physical Examination:
Body Weight: 4.2 kg
General: Quiet, alert, and responsive. Ambulatory on all four
limbs. T = 104.3°F (40.2°C), HR = 180 beats/min, RR = 30
breaths/min, mucous membranes pink, CRT = 1 s.
Integument: A dull haircoat was present. There was no
evidence of ectoparasites.
Eyes: Bilateral anterior uveitis was present; fundoscopic
examination was difficult due to aqueous flare.
Musculoskeletal: Body condition score was 4/9; possible
discomfort was detected on muscle palpation.
Gastrointestinal: The cranial abdomen was tense with
abdominal splinting noted on palpation.
All Other Systems: No significant abnormalities were noted.
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Laboratory Findings:
Complete Blood Count: All values were within reference
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53. Pfohl JC, Dewey CW. Intracranial Toxoplasma gondii granuloma
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55. Alves L, Gorgas D, Vandevelde M, et  al. Segmental meningomy-
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65. Lappin MR, George JW, Pedersen NC, et al. Primary and second-
ary Toxoplasma gondii infection in normal and feline immunodefi-
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72. Lappin M. Unpublished observations, 2012.
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CHAPTER 73
Neosporosis
Jane E. Sykes
Overview of Neosporosis
First Described: 1984, in boxer dogs in Norway (Bjerkas and
others)1
Cause: Neospora caninum, a coccidial protozoan parasite
(phylum Apicomplexa)
Affected Hosts: Dogs are the definitive hosts and are the only
species known to complete the sexual phase of N. cani-
num replication with passage of oocysts in the feces. Clini-
cal illness primarily occurs in dogs and cattle, but sheep,
deer, and water buffalo may also act as intermediate hosts.
Geographic Distribution: Worldwide
Mode of Transmission: Dogs can be infected transplacentally
or through the transmammary route. Postnatally, dogs
may be infected after they ingest infected tissues of inter-
mediate hosts or bovine fetal membranes
Major Clinical Signs: Ascending paralysis, muscle atrophy,
neurologic (especially cerebellar) signs, nodular dermati-
tis, respiratory distress
Differential Diagnoses: Toxoplasmosis, sarcocystosis, hepa-
tozoonosis, deep mycoses, protothecosis, rabies, canine
distemper virus infection, West Nile virus infection, canine
monocytic ehrlichiosis, migrating ascarid infections, neu-
ronal storage diseases, granulomatous meningoencepha-
litis, immune-mediated neuromuscular disease, primary
or metastatic neoplasia
Human Health Significance: There is no clear evidence that
N. caninum infects humans.
Etiology and Epidemiology
Neospora caninum is an obligately intracellular protozoan
pathogen that closely resembles Toxoplasma gondii; before its
description in 1984, it was misidentified as T. gondii. In contrast
to T. gondii, domestic dogs and wild canids (coyotes, dingos,
and probably wolves), and not cats, act as definitive hosts of
N. caninum and shed oocysts after ingestion of N. caninum tis-
sue cysts (enteroepithelial cycle) (Figure 73-1). Clinical neospo-
rosis results from systemic replication of N. caninum tachyzoites
(which divide by a process known as endodyogeny) and has
been described in dogs and certain domestic and wild herbivores
such as cattle, sheep, goats, deer, and marsupials. A related para-
site, Neospora hughesi, sporadically causes myeloencephalitis in
horses.2 This organism has an unknown definitive host that is
unlikely to be the dog, based on experimental infection studies.3
704
N. caninum is one of the most important causes of infertility,
abortion, and neonatal mortality in cattle worldwide. Sheep can
also develop reproductive and neonatal disease, but the eco-
nomic importance of N. caninum in sheep is less clear when
compared with cattle.4 Herbivores likely become infected when
they ingest oocysts shed in canine feces or through subclinical
transplacental transmission. Oocysts sporulate and become
infective 24 to 72 hours after they are shed in canine feces and
can survive in the environment for prolonged periods. After
oocysts are ingested, sporozoites are released in the intestinal
tract and penetrate intestinal epithelial cells. Organisms can then
disseminate to a variety of tissues, where they ultimately encyst
as bradyzoite cysts. In dogs, pregnancy is associated with reac-
tivation of bradyzoite cysts, with replication of tachyzoites and
subsequent abortion or transplacental transmission. In breeding
dogs, repeated reactivation of N. caninum replication occurs in
late gestation with successive pregnancies, so that multiple litters
of puppies can be affected. Transmission may also occur via the
transmammary route. Infection of immunologically naïve cattle
late in pregnancy is most likely to lead to transplacental spread
with abortion (also known as exogenous transplacental trans-
mission).5 However, transplacental transmission as a result of
reactivation of latent infection during pregnancy (endogenous
transplacental transmission) can also occur.6 The pregnant cow
typically shows no other overt clinical signs of infection. Although
antibodies to N. caninum have been found in cats and humans,
naturally occurring neosporosis has not been described in these
species.
Aside from transplacental transmission, dogs can become
infected and shed oocysts when they ingest bovine placen-
tal material as well as other bovine tissues, such as muscle,
liver, brain, and heart muscle.7 This horizontal transmission
is important and is thought to maintain infection in the dog
population.8 The DNA of N. caninum has been found in birds,
small rodents, and lagomorphs, but whether predation of these
species leads to oocyst shedding by dogs is not clear.4 Experi-
mental evidence suggests that ingestion of infected chicken
eggs by dogs may lead to oocyst shedding.9 However, inges-
tion of oocysts by dogs does not lead to oocyst shedding or
development of tissue cysts.10 When dogs do shed oocysts,
the shedding begins 5 to 13 days after infection, and only
low numbers of oocysts are shed for a short period (several
days); seroconversion does not always occur. Rarely, pro-
tracted oocyst shedding (several months) by dogs has been
described.11 Puppies shed more oocysts than adult dogs,12 and
the number of oocysts shed decreases with repeated exposures
to bradyzoite cysts.
In dogs, seroprevalence increases with age (because of an
increased likelihood of exposure through horizontal transmis-
sion over time) and is higher in free-roaming dogs than in pet
 CHAPTER 73  Neosporosis 705

FIGURE 73-1  Life cycle of Neospora caninum. Dogs become infected when they ingest tissue cysts in bovine placental material and other bovine tissues. Bradyzoites are released in
the intestine and transform into merozoites, where they undergo merogony. A zygote forms and is shed in the feces as an unsporulated oocyst. In addition, organisms can penetrate the
dog’s intestinal tract and form tissue cysts. Reactivation of these cysts in pregnancy can result in repeated transplacental transmission to the fetus. Herbivores are infected when they ingest
sporulated oocysts. Infection of cattle can lead to transplacental spread of tachyzoites and abortion.

dogs. Dogs that reside on cattle farms or dogs in rural areas
are also more likely to be seropositive than dogs that reside in
urban areas.13-16 Introduction of infected dogs to a farm can
lead to outbreaks of abortion in cattle on the farm,17 and the
more dogs present on a farm, the greater the chance that cattle
will be exposed. Although mixed breed dogs are more likely to
be seropositive (exposed) than purebred dogs, purebred dogs
often develop clinical neosporosis,18 possibly because of genetic
defects in cell-mediated immune function. Commonly affected
breeds in the literature and/or in the author’s practice include
boxers, Rhodesian ridgebacks, bull mastiffs, greyhounds, basset
hounds, Labrador retrievers, Rottweilers, and West Highland
white terriers.
Clinical Features
Signs and Their Pathogenesis
Although exposure to N. caninum is common, clinical disease
is uncommon in dogs. The development of clinical neosporo-
sis, the signs that develop, and their rate of progression may
depend on the virulence of the infecting N. caninum strain and
the age and immunocompetence of the host.4,19 Many affected
dogs are infected transplacentally, after which clinical signs
usually become apparent from 4 weeks to 6 months of age. Up
to 50% of puppies in a litter can be infected, but not all puppies
develop signs simultaneously, and sometimes only one puppy
in the litter develops clinical illness.20 Stillbirths and neonatal
deaths may also be reported.18 Reactivation of subclinical infec-
tion can occur in older dogs after treatment with immunosup-
pressive drugs or chemotherapy.21-23 Horizontal acquisition of
infection by adult dogs may occur through consumption of raw
meat; one dog that developed neosporosis had a history of being
fed raw elk meat.22
Transplacental infection of puppies usually leads to myositis
and polyradiculoneuritis that initially involves the lumbosacral spi-
nal nerve roots. This is manifested as ascending paralysis, muscle
atrophy, and fibrous muscle contracture with arthrogryposis (joint
contracture), hyperextension of the pelvic limbs, and loss of patel-
lar reflexes.18 Early signs of disease include exercise intolerance,
ataxia, a high-stepping gait, splaying of the pelvic limbs, a “bunny
hopping” gait, urinary incontinence, and/or muscle pain.18,24-26
Clinical signs progress in some dogs to lethargy, tetraplegia, inabil-
ity to fully open the mouth, dysphagia, and respiratory difficulty.
Involvement of esophageal muscle may lead to megaesophagus.27,28
Mandibular paralysis, vomiting, and lethargy were described as the
only clinical signs in one affected dog.28 Although ocular lesions
are not a major clinical feature of neosporosis, focal chorioretini-
tis has been reported in some puppies.29 Less often, young dogs
develop signs of hepatitis; pneumonia; meningoencephalomyelitis;
or myocarditis with arrhythmias, acute onset of respiratory dis-
tress, or sudden death.26,27,30,31 Puppies with neuromuscular signs
may have subclinical infection of other organ systems.27
In contrast to puppies, adult dogs typically develop polymy-
ositis and/or meningoencephalomyelitis, with a variety of neu-
rologic signs.19,27,32 Although lesions can occur throughout the
central nervous system (CNS),27 N. caninum has a predilection
for the cerebellum.19 Some dogs can develop cerebellar atro-
phy.19,33 Disseminated infections that involve the myocardium,
liver, pancreas, lungs, skin, and/or eyes can also occur in adult
dogs but are less common than encephalitis. In one unusual
case, N. caninum tachyzoites were found in the peritoneal fluid
of a dog with bacterial peritonitis.34
Physical Examination Findings
Physical examination findings in puppies with polyradiculoneuritis-
myositis vary from mild ataxia and muscle atrophy to
706 SECTION 4  Protozoal Diseases

TABLE 73-1
Diagnostic Assays Available for Neosporosis in Dogs
Assay Specimen Type Target Performance
Fecal flotation Feces N. caninum oocysts Dogs with clinical neosporosis typically
do not shed oocysts so this has a very
low sensitivity and is not used routinely
for diagnosis. Oocysts cannot be readily
distinguished from those of Hammondia
heydorni.
Fecal PCR assay Feces N. caninum DNA As for fecal flotation.
Cytologic examination CSF, aspirates of skin N. caninum tachyzoites Insensitive except in the case of skin lesions.
lesions, rarely specimens Confirms active infection and is generally
from the respiratory tract associated with disease. Organisms cannot
be readily distinguished from Toxoplasma
gondii.
Serology (IFA or ELISA) Serum or CSF Antibodies to N. caninum Assays can vary in performance between
laboratories. A fourfold increase in titer is
associated with recent infection. Dogs with
chronic neosporosis may not show a rise in
titer, but titers >1:800 in conjunction with
consistent clinical signs suggest neosporosis.
Histopathology Muscle biopsies, multiple N. caninum bradyzoites If organisms are detected in the presence
tissues collected at nec- and tachyzoites of inflammation and necrosis, clinical
ropsy neosporosis is likely. May be difficult to
differentiate N. caninum and T. gondii.
Immunohistochemistry can aid in the
differentiation of the two species.
PCR assay Muscle or skin biopsies, N. caninum DNA Assay performance may vary between
body fluids (especially laboratories. Because positive PCR results
CSF), tissues collected at can occur in healthy dogs, they must be
necropsy interpreted in light of clinical signs and
other findings in the specimens tested.

tetraparesis with rigid extension of one or both pelvic limbs
and arthrogryposis. Reduced patellar and sometimes other seg-
mental reflexes (including the perianal reflex) are often pres-
ent.18,24,35,36 Severely affected dogs may be lethargic and exhibit
respiratory difficulty, and it may only be possible to open the
mouth a few centimeters. Fever is rare.18
Adult dogs with N. caninum meningoencephalitis may be
obtunded and exhibit signs that include ataxia, circling, head tilt,
nystagmus, hypermetria, head tremors, delayed placing reactions,
abnormal cranial nerve function, anisocoria, depressed segmental
reflexes, tetraparesis, cervical hyperesthesia, and seizures.
Dogs with myocardial involvement may develop acute onset
of respiratory difficulty and cyanosis.18,27 Cutaneous involvement
is manifested as dermatitis with single or multifocal firm nodules,
crusting, and/or ulceration, which can appear at additional sites
over time and may or may not be accompanied by other systemic
signs of infection.21,37-40 Rarely, dogs with pulmonary involvement
are evaluated for cough or respiratory difficulty.41
especially cerebellar) signs or nodular dermatitis, especially in
predisposed breeds or dogs treated with immunosuppressive
drugs. Diagnosis most often relies on serology or detection of
protozoal organisms in body fluids, aspirates, or tissues using
light microscopy and/or PCR assays (Table 73-1). Often, ante-
mortem diagnosis is based only on serologic testing because
organisms are not easily found.
Laboratory Abnormalities
Complete Blood Count, Serum Biochemistry Panel, and Urinalysis
As for toxoplasmosis, there are no specific hematologic or bio-
chemical abnormalities that suggest neosporosis. The CBC is
often normal, but occasionally mild nonregenerative anemia,
mild eosinophilia, or monocytosis is evident.18,32 The serum
biochemistry panel reveals increased creatine kinase activity in
dogs with myositis.19,23,24,26 Occasionally, increased activity of
serum ALT (due to hepatitis or severe myositis) or hyperglobu-
linemia is evident.23,32 The urinalysis is generally unremarkable.

Diagnosis Cerebrospinal Fluid Analysis

The CSF of dogs with N. caninum meningoencephalitis may
Neosporosis should be suspected in young dogs (<1 year of be normal or show mild to markedly increased total nuclear
age) that develop ascending paralysis and muscle atrophy. It cell count (TNCC) (sometimes >1000 cells/µL) and mild to
should also be suspected in dogs that develop neurologic (and markedly increased CSF protein concentration.19,32 Usually,
 CHAPTER 73  Neosporosis 707

a mononuclear pleocytosis is present, but occasionally the

response is mixed or primarily composed of neutrophils or
eosinophils.19,24,32,35 N. caninum tachyzoites are rarely seen in
CSF.22,32

Diagnostic Imaging
Plain Radiography
Thoracic radiographs in dogs with neosporosis are usually unre-
markable but may show a diffuse interstitial pattern in dogs
with pneumonia, or evidence of megaesophagus in dogs with
neuromuscular signs.18

Sonographic Findings
Abnormalities are rarely detected on abdominal ultrasound
examination of dogs with neosporosis.

Advanced Imaging
MRI of adult dogs with cerebellar signs associated with
N. caninum infection may show moderate to marked bilateral
cerebellar atrophy.19 Meningeal contrast enhancement may be
present.19,32 Single or multifocal T2-weighted and FLAIR hyper-
intensities that enhance with contrast on T1-weighted images
may be present within the cerebellum or other parts of the brain FIGURE 73-2  Sagittal T1-weighted postcontrast MRI image from a 4-year-old
(Figure 73-2). Mild to moderate heterogenous T2-weighted and female spayed Chihuahua with suspected Neospora caninum meningoencephalitis. Mul-
FLAIR hyperintensities can also be seen in the masseter and tiple poorly defined regions of contrast enhancement were identified in the cerebral
temporalis muscles, which may appear atrophied. cortices (large arrow), and mild meningeal enhancement was evident (small arrow). CSF
analysis revealed a mononuclear pleocytosis, and Neospora DNA was detected using PCR
Electrodiagnostic Testing in the cerebrospinal fluid.
Electromyography in dogs with neosporosis may reveal sponta-
neous fibrillation potentials, positive sharp waves, and repetitive
discharges.24,26 In some dogs, nerve conduction studies have
revealed reduced conduction velocities and compound muscle
action potentials.24

Microbiologic Tests
Cytologic Examination
The tachyzoites of N. caninum may be visualized within aspirates
of skin lesions (which often contain many organisms) and rarely
in CSF or other body fluids. They are approximately 6 × 1 µm
and oval to crescent-shaped.22,32,34,37 They may be seen singly,
in pairs, or in larger groups, extracellularly and intracellularly,
and are often associated with a pyogranulomatous inflamma-
tory response (Figure 73-3). In the dog, N. caninum tachyzoites
cannot be distinguished from those of T. gondii on cytologic
examination. Because some cross-reactivity between N. caninum
and T. gondii can occur with immunocytochemistry depending
on the antibodies used, the use of PCR assays is recommended
to definitively identify organisms as N. caninum. For unknown
reasons, such cross-reactivity may be most likely to occur in dermal
neosporosis.8
FIGURE 73-3  Cytology of abdominal fluid sediment in a 7-year old male neutered
Fecal Flotation Rhodesian ridgeback showing intracellular and extracellular Neospora caninum tachyzo-
Fecal flotation with sugar or zinc sulfate solutions can be used ites (arrows). Nucleated cells are predominantly nondegenerate to mildly degenerate
to detect the oocysts of N. caninum in feces. However, clinically neutrophils.
affected dogs do not typically shed oocysts. When oocysts are
seen, they are indistinguishable from those of Hammondia hey- are widely used, but ELISA assays have also been developed and
dorni or Toxoplasma gondii. may be used to detect IgM and IgG antibody. Seroconversion
usually occurs within 2 to 3 weeks of infection, and a rising
Serologic Diagnosis serum titer can be identified in acutely infected dogs.26 This rise
Serologic assays are available commercially that detect antibody may not occur in chronically infected dogs, so in this case a single
to N. caninum in serum and/or in CSF. Titers are often higher high titer in a dog with consistent clinical signs is suggestive
in serum than in the CSF of affected dogs. Indirect IFA assays of the diagnosis. Some dogs with neosporosis have extremely
708 SECTION 4  Protozoal Diseases
A B
FIGURE 73-4  Histopathology of the cerebellum from a 6-year-old intact male Rhodesian ridgeback with neosporosis. A, Large areas of malacia were present admixed with a marked
granulomatous inflammatory infiltrate and abundant protozoal cysts (arrowheads). B, The protozoal cysts had a thick wall and stained positive with anti-Neospora antibodies (not shown).
(Courtesy Dr. Robert Higgins, University of California, Davis Veterinary Anatomic Pathology Service.)
high serum titers (e.g., in the tens of thousands), but others have
only low antibody titers. The latter situation might reflect the
presence of underlying immunodeficiency and inability to pro-
duce antibodies. Because interassay variation may cause results
to differ between laboratories,42 the same laboratory should
always be used when paired titers are performed or when titers
are monitored over time.
Some weak serologic cross-reactivity may occur between the
antigens of T. gondii and N. caninum, so low titers to T. gon-
dii may be present in dogs infected with N. caninum. Cross-­
reactivity appears to be less common when IFA assays are
used.42 Serologic cross-reactivity can also occur to N. hughesi,
again at a slightly lower titer,43 but the extent to which this
organism infects dogs is not clear.
Molecular Diagnosis Using the Polymerase Chain Reaction
Both conventional and real-time PCR assays have been devel-
oped for diagnosis of neosporosis.44-46 Some are available for
diagnosis on a commercial basis from veterinary diagnostic
laboratories. Suitable specimens for testing include CSF, tissue
biopsies (e.g., muscle), or tissues collected at necropsy. The
sensitivity and specificity of these assays has the potential to
vary considerably between laboratories, and more informa-
tion is required on how these assays perform for antemortem
diagnosis of clinical neosporosis in dogs. Because N. caninum
DNA can be detected in tissues from healthy dogs, PCR results
must always be interpreted in light of the clinicopathologic
findings and serologic test results. In one study, N. caninum
DNA was detected in the liver and/or spleen of one third of
shelter dogs that had been euthanized, and PCR assay results
did not always correlate with the results of serology.44 PCR
assays can be used to confirm that organisms seen in tissues or
body fluids are truly N. caninum and not another protozoan
parasite such as Sarcocystis or T. gondii.23
Pathologic Findings
Gross pathologic findings may be absent in dogs with neospo-
rosis, or foci of discoloration may be present in the brain and
spinal cord, and/or there may be evidence of meningitis (e.g.,
meningeal thickening or adherence of the meninges to the brain
or cranial vault). White streaks within muscle may be found in
affected puppies.24 Histopathology in these puppies reveals poly-
radiculoneuritis with myelin loss and axonal degeneration, and
there may be evidence of muscle fiber degeneration and fibrosis.
Examination of the brain and spinal cord of dogs with
CNS involvement usually reveals multifocal nonsuppurative
meningoencephalitis and/or myelitis with neuronal degenera-
tion and necrosis, perivascular cuffing, and gliosis. Both gray
and white matter can be affected, and the cerebellum may be
primarily involved. A mixed inflammatory response with myo-
necrosis and sometimes dystrophic mineralization may be seen
in affected skeletal or cardiac muscle. Bradyzoite cysts and/or
tachyzoites may be evident (Figure 73-4). Bradyzoite cysts are
up to 100 µm in diameter and have a wall that is 4 µm in diam-
eter, which is thicker than that for T. gondii. Skin biopsies from
dogs with dermal neosporosis contain tachyzoites and pyogran-
ulomatous inflammation. Occasionally (such as in acute, ful-
minant infections in immunosuppressed dogs), tachyzoites are
found in other tissues, including the peripheral nerves, the eye,
reproductive tissues, adrenal glands, lung, liver, kidney, spleen,
and esophageal muscle.27 If intralesional protozoa are identi-
fied, immunohistochemistry and PCR assays can be used to dis-
tinguish N. caninum from other protozoal species.40 Although
serologic cross-reactivity between N. caninum and T. gondii is
uncommon, it can occur with some antibodies, so immunohis-
tochemistry is best performed with a panel of antibodies to each
protozoan parasite.
In healthy dogs or dogs that die of other diseases, rare brady-
zoite cysts in the absence of an inflammatory response may be
found at necropsy in skeletal muscle and the CNS.
Treatment and Prognosis
The primary antimicrobial drug that has been used to treat neo-
sporosis in dogs is clindamycin (Table 73-2). Treatment is usually
only temporarily, partially, or completely ineffective, and long
periods (>8 weeks) of treatment may be required.18,19,39 Bradyzo-
ite cysts persist despite treatment with clindamycin.47 Treatment
appears to be most effective for cutaneous neosporosis, although
one dog developed neurologic signs when treatment was discontin-
ued, such that reinstitution of long-term treatment was needed.39
Other treatments that have been successful (or partially successful)

TABLE 73-2
Antimicrobials Used to Treat Neosporosis in Dogs
Drug Dose (mg/kg) Route
Clindamycin 10-12 mg/kg PO
Trimethoprim-sulfa 15 mg/kg PO
Pyrimethamine 1 mg/kg PO
Ponazuril 20 mg/kg PO
are trimethoprim-sulfadiazine-pyrimethamine, combinations of
clindamycin and trimethoprim-sulfadiazine, or combinations of
clindamycin and pyrimethamine. The efficacy of ponazuril for
treatment of canine neosporosis requires further study but shows
promise based on studies in mice and cattle.8 Treatment should be
continued so long as clinical improvement is occurring. The prog-
nosis for puppies once muscle contracture has developed is poor.
It has been suggested that once neosporosis has been diagnosed
in a puppy, all dogs in a litter should be treated, because the later
treatment is initiated after the onset of clinical signs, the worse
the prognosis.48 At the minimum, puppies whose littermates have
succumbed to neosporosis should have their serum antibody titer
to N. caninum checked, and treatment should be considered for
those with positive titers. The use of immunosuppressive drugs
should be avoided in seropositive puppies.
A low dose of glucocorticoids (e.g., prednisone, 0.5 mg/kg PO
q12h) in addition to antiprotozoals is often used to treat N. cani-
num meningoencephalomyelitis, but no prospective controlled
trials have been performed to determine if this alters outcome. Very
high doses of methylprednisolone acetate reactivate experimental
N. caninum infections in dogs.49 Physiotherapy may also be
beneficial for young dogs with polyradiculoneuritis-myositis.
Immunity and Vaccination
Immunity to N. caninum is dependent on cell mediated immune
responses. No vaccines for canine neosporosis exist.
CASE EXAMPLE
Signalment: “Lenny,” a 6-year-old intact male Rhodesian
ridgeback from coastal northern California
History: Lenny was evaluated on a referral basis for a 1-week
history of an abnormal gait. The owner first noticed that
Lenny was not paying attention during an obedience
show. Over the next few days Lenny was observed to
stumble on stairs, and he also fell from a bed. For the
3 days before evaluation the owner had noted that Lenny
appeared uncoordinated and off balance. There had been no
changes in his mentation and no evidence of inappetence,
vomiting, diarrhea, or changes in thirst or urination. A soft,
intermittent nonproductive cough had been noted since
the onset of the other clinical signs. Bloodwork at a local
veterinary clinic showed a normal CBC, increased activity of
serum creatine kinase (2922 U/L, reference range [RR] 10-200
CHAPTER 73  Neosporosis 709
Interval (hours) Duration (days)
q8h At least 4 weeks
q12h At least 4 weeks
q24h At least 4 weeks
q24h At least 4 weeks
Prevention
Neosporosis on farms can be prevented by not allowing dogs
to access bovine placental materials, dead calves, or raw or
undercooked meat and by preventing dogs from defecating
around livestock. Predation should also be discouraged. The
use of a muzzle for dogs that work on farms has been sug-
gested to prevent Neospora abortions in cattle.8 It may be
necessary to stop the breeding of bitches that have puppies
that develop neosporosis, and the owners of puppies that
are diagnosed with neosporosis should be advised to inform
the breeder of the diagnosis. No drugs are known to prevent
transplacental transmission. Where possible, avoidance of
excessive pharmacologic immunosuppression (e.g., with glu-
cocorticoids, cyclosporine, azathioprine, or other immuno-
suppressive drugs) may help to prevent the disease in adult
dogs. However, the relative rarity of reactivated neosporosis
in adult dogs does not justify routine serologic screening of
dogs for infection before commencing immunosuppressive
drug treatment.
Public Health Aspects
Although antibodies to N. caninum have been detected in the
serum of humans, the DNA of N. caninum has never been
found in humans, and currently N. caninum is not thought to
be a zoonotic risk.
U/L), ALT (155 U/L, RR 5-60 U/L), and AST (135 U/L, RR 5-55
U/L). Serum thyroxine concentration was within the reference
range. A urinalysis was unremarkable and aerobic bacterial
urine culture was negative. Serology for antibodies to Borrelia
burgdorferi, Anaplasma spp., Ehrlichia canis, and antigen of
Dirofilaria immitis (4Dx SNAP test, IDEXX Laboratories) was
negative. A T. gondii titer was pending. Lenny was treated
with clindamycin (7.5 mg/kg PO q12h) and doxycycline (5
mg/kg PO q12h) with no signs of improvement.
Lenny had traveled extensively to Iowa, Colorado, and Kentucky
but had no history of toxin exposure or trauma. He was fed
a raw diet that consisted of turkey necks, chicken meat,
lamb, and vegetables. He was up to date on routine vacci-
nations (distemper, adenovirus, parvovirus, and rabies) and
on heartworm prophylaxis, but did not receive flea or tick
prophylaxis and had been exposed to ticks in the past. He
had been bred once and only one of three puppies survived.
There was no other significant medical history.
710 SECTION 4  Protozoal Diseases
Physical Examination:
Body Weight: 40.2 kg.
General: Bright and alert, hydrated. T = 100.5°F (38.1°C),
HR = 80 beats/min, panting, mucous membranes pink and
moist, CRT 1 s.
Musculoskeletal: Body condition score was 4/9. Truncal ataxia
was present with a wide-based stance (see Neurologic
Examination). There was no evidence of lameness or muscle
atrophy.
All Other Systems: With the exception of the neurologic
examination, no other clinically significant abnormalities
were detected.
Neurologic Examination: The dog was alert and responsive.
Truncal ataxia was present with hypermetria that was most
pronounced on the left side. Head tremors were also noted.
A slight left-sided head tilt was present, together with a
decreased to absent menace response on the left side.
There was normal physiologic nystagmus and no evidence
of spontaneous nystagmus. Delayed placing reactions
were detected in all four limbs but were most pronounced
on the right side. Spinal reflexes were within normal
limits. Neuroanatomic location: cerebellar/vestibular
disease.
Laboratory Findings: Muscle biochemistry panel: AST 85
U/L (21-54 U/L), creatine kinase 1683 U/L (46-320 U/L).
Imaging Findings:
Thoracic Radiographs and Abdominal Ultrasound
Examination: No abnormalities were detected.
Magnetic Resonance Imaging (Brain): On T2-weighted
images, there was increased intensity of the vermis and
left cerebellar hemisphere. The cerebellum appeared
normal on T1-weighted images. Subtle regions of contrast
enhancement were suspected in the left cerebellum on
sagittal T1-weighted postcontrast images.
Other Testing: CSF analysis: Total nucleated cell count was
62 cells/µL (normal <2 cells/µL); total protein was 75 mg/dL
(normal <25 mg/dL). There were 86% eosinophils, 8% small
mononuclear cells, 5% large mononuclear cells, and 1%
nondegenerate neutrophils. The large mononuclear cells were
mostly of unremarkable morphology but occasionally had
mildly increased cytoplasmic vacuolation. No infectious agents
were seen.
Microbiologic Testing: Serologic testing (IFA): Neospora
caninum serum antibody titer positive at 1:20,480; the CSF
titer was 1:640.
Zinc sulfate fecal flotation: Negative for parasite ova.
Fecal Baermann examination: Negative for parasite larvae.
Treatment: Before the Neospora titers were available, a
diagnosis of eosinophilic meningoencephalitis was made,
and the dog was treated with prednisone (0.5 mg/kg
PO q12h) but showed no clinical improvement over the
subsequent 3 days. A second CSF specimen collected at
that time had a cell count of 37 cells/µL and a total protein
concentration of 59 mg/dL, with no change in the differential
cell count. Lenny was discharged from the hospital, and once
the N. caninum serology results became available (1 week
later), treatment with trimethoprim-sulfamethoxazole (15
mg/kg PO q12h) and ponazuril (10 mg/kg, or 2.5 mL of 15%
ponazuril paste PO q24h) was added. A baseline Schirmer
tear test (performed to monitor for keratoconjunctivitis
sicca secondary to sulfamethoxazole) was negative. At a
recheck examination 3 weeks later, all neurologic signs
were persistent but mildly improved. A CBC showed mild,
nonregenerative anemia (34.9%) and mild bandemia (198
cells/µL). Serum CK activity was 88 U/L. CSF analysis showed
a total protein concentration of 33 mg/dL and a TNCC of
2 cells/µL, with 0% eosinophils, 51% small mononuclear
cells, and 49% mildly reactive large mononuclear cells. The
serum and CSF antibody titers to N. caninum were both
1:5120. A Schirmer tear test was normal. Two weeks later,
the trimethoprim-sulfamethoxazole was discontinued.
Neurologic signs persisted but continued to show mild but
slow improvement over the next 3 weeks. The ponazuril
was discontinued, but after an additional 3 weeks, the head
tremors worsened slightly. Blood work was unchanged, CSF
analysis (cisternal and lumbar) was within normal limits, and
serum and CSF N. caninum antibody titers were 1:1280 and
1:640, respectively. The prednisone was also discontinued.
Two months later (4 months after initial diagnosis), severe
ataxia and falling returned over a 2-week period. The owner
declined reinstitution of antiprotozoal treatment and
elected euthanasia. A complete necropsy revealed severe,
locally extensive, necrotizing granulomatous encephalitis
that was localized to the cerebellum. Abundant protozoal
cysts were present within the cerebellar lesions, which were
identified using immunohistochemical stains as N. caninum.
Protozoa were not visualized in any other tissues.
Diagnosis: Granulomatous cerebellitis secondary to
N. caninum infection.
Comments: Neosporosis in this dog resulted from cerebellar
encephalitis, which illustrated the suggested predilection
of N. caninum for the cerebellum. Development of
encephalitis rather than polyradiculoneuritis-myositis is
typical of neosporosis in adult dogs. It is possible this dog
contracted neosporosis as a result of consumption of a raw
food diet; tissue cysts have been identified in CNS tissues of
a lamb,50 and chickens are suspected as intermediate hosts
based on seropositivity and positive PCR assay results on
brain tissue.51 The inflammatory response in the CSF was
profoundly eosinophilic, as previously reported in other
dogs with neosporosis. Fecal flotation was negative for
N. caninum oocysts, which is expected given the life cycle
of the parasite and the short shedding period with low
numbers of oocysts shed. The fecal examinations were
performed to evaluate for the possibility of visceral larva
migrans, given the CSF eosinophilia. Although treatment
with trimethoprim-sulfamethoxazole and then ponazuril
led to resolution of CSF abnormalities, clinical signs
showed only mild improvement. Treatment of neosporosis
with glucocorticoids remains controversial, but attempts
to reduce the prednisone dose were associated with
worsening of clinical signs. Ultimately, all treatment was
discontinued because of minimal improvement in clinical
signs but resolution of laboratory abnormalities. Relapse
subsequently ensued, and the diagnosis was confirmed at
necropsy.

SUGGESTED READINGS
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Dubey JP, Schares G. Neosporosis in animals—the last five years. Vet
Parasitol. 2011;180:90-108.
Garosi L, Dawson A, Couturier J, et  al. Necrotizing cerebellitis and
cerebellar atrophy caused by Neospora caninum infection: magnetic
resonance imaging and clinicopathologic findings in seven dogs. J Vet
Intern Med. 2010;24:571-578.
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CHAPTER 74
Leishmaniosis
Jane E. Sykes, Gad Baneth, and Christine A. Petersen
Overview of Leishmaniosis
First Described: The cause of visceral leishmaniosis was first
described in India in 1903 (separately by William Leishman
and Charles Donovan)1
Causes: Canine and feline visceral leishmaniosis is caused by
Leishmania infantum (syn. L. chagasi) (phylum Euglenozoa,
class Kinetoplasta, family Trypanosomatidae). Other spe-
cies such as L. braziliensis cause localized cutaneous lesions
in South America (American tegumentary leishmaniosis).
Affected Hosts: Dogs and less commonly cats; also humans,
rodents, wild canids, and marsupial species
Geographic Distribution: Primarily southern Europe and the
Middle East, central and South America; also some parts
of the United States. Travel-related disease can occur in
non-endemic regions worldwide.
Mode of Transmission: Phlebotomus and Lutzomyia sandflies.
Transmission through blood transfusion as well as vertical
transmission can occur.
Major Clinical Signs: Weight loss, inappetence, scaling
and/or ulcerative cutaneous lesions, onychogryphosis,
fever, keratoconjunctivitis, uveitis, lymphadenopathy,
hepatosplenomegaly, pallor, lameness, signs of renal
failure
Differential Diagnoses: Differential diagnoses in dogs with
visceral disease include canine monocytic ehrlichio-
sis, babesiosis, histoplasmosis, brucellosis, hemic
neoplasia, and primary autoimmune diseases such as
systemic lupus erythematosus. Differential diagnoses
for cutaneous lesions in dogs include demodectic or
sarcoptic mange, pyoderma, Malassezia dermatitis,
cutaneous vasculitis, and pemphigus foliaceus. Differ-
ential diagnoses for cutaneous lesions in cats include
squamous cell carcinoma, cutaneous vasculitis, her-
pesviral dermatitis, and dermatophytosis. The primary
differential for American tegumentary leishmaniasis is
sporotrichosis.
Human Health Significance: Human infections are primarily
transmitted by sandflies and involve a large number of dif-
ferent Leishmania species. Dogs are considered the major
reservoir for L. infantum infections. Precautions should
be taken when handling potentially infected tissue and
blood specimens.
CANINE LEISHMANIOSIS
Etiology and Epidemiology
The leishmaniases are a group of zoonotic vector-borne diseases
caused by various species of the protozoa Leishmania, which are
primarily transmitted by sandflies. Around 30 species of Leish-
mania exist worldwide, and at least 20 species cause disease in
humans, although the taxonomy of Leishmania spp. has been
highly debated.2,3 There are two subgenera of Leishmania, Leish-
mania and Vianna, which develop in the foregut or the hindgut/
midgut of the sandfly vector, respectively. Human and animal dis-
ease due to Leishmania spp. is distributed across five continents
(Africa, Asia, Europe, and the Americas) and nearly 100 countries
(Figure 74-1). Disease syndromes in humans are divided into cuta-
neous leishmaniasis (CL), mucosal leishmaniasis (ML), and visceral
leishmaniasis (VL). Disease syndromes in dogs are divided into
visceral leishmaniosis and American tegumentary leishmaniosis, a
localized cutaneous form that occurs in parts of South America.
The term ‘leishmaniosis’ as opposed to ‘leishmaniasis’ is preferred
to distinguish the disease in animals from that in humans.
Leishmania species are transmitted by female sandflies of
the genus Phlebotomus in the Old World (Africa, Asia, and
Europe) and Lutzomyia in the New World (i.e., the Americas).
The regional prevalence of leishmaniasis, and the type of disease
syndrome that develops, varies with the infecting Leishmania
species present, sandfly species in the region, and host responses
to infection. Some sandfly species transmit certain Leishmania
species primarily to humans (anthroponotic leishmaniasis),
whereas other sandfly species transmit Leishmania species to
mammalian host species, such as dogs, cats, and rodents. Dogs
and rodents are important reservoirs for Leishmania infection of
humans (zoonotic leishmaniasis). Thus, the major mammalian
reservoirs for the disease in a region depend on the Leishmania
species and the sandfly species present.
The life cycle of Leishmania alternates between two major
forms: the promastigote and the amastigote (Figure 74-2). The
promastigote resides in the gut of the sandfly vector and is an
elongated, flagellated form. Promastigotes are inoculated into
tissues when the sandfly feeds, where they are phagocytized
by macrophages in the dermis and transform into intracellu-
lar, nonmotile, ovoid, or round amastigotes. The amastigotes
survive and replicate within macrophage phagolysosomes, and
eventually the infected macrophages rupture. Released amasti-
gotes then infect other macrophages that have been attracted
to the site of infection. If the infected host fails to control the
infection, amastigotes disseminate via regional lymphatics and
the blood to infect the entire reticuloendothelial system. Intra-
cellular amastigotes that are ingested by a female sandfly during
713
714 SECTION 4  Protozoal Diseases
FIGURE 74-1  Geographic distribution of visceral leishmaniases in humans and dogs.
FIGURE 74-2  Life cycle of Leishmania infantum. Dogs, cats, and humans become
infected when they are bitten by a sandfly. Promastigotes are inoculated into the der-
mis and penetrate macrophages. Within the macrophage, they transform into amas-
tigotes and replicate within a phagolysosome. The macrophage ruptures and new
macrophages are infected. If the host fails to control the infection in the skin, amasti-
gotes disseminate via regional lymphatics and the blood to infect the entire reticulo-
endothelial system. Amastigotes that are ingested by a female sandfly during feeding
convert back into promastigotes, and replication of promastigotes within the sandfly
completes the life cycle.
feeding convert back into promastigotes over a period of about
1 week, and replication of promastigotes within the sandfly
completes the life cycle. Sandflies breed in cracks in the walls
of dwellings, rubble, and in rodent burrows, feed primarily at
night, and fly a maximum of 2.5 km from their breeding sites.
As a result, the prevalence of infection in dogs can vary consid-
erably between adjacent regions. Lesser modes of transmission
of Leishmania spp. include vertical transmission from bitches to
puppies, venereal transmission, and blood transfusion.4-7 These
modes of transmission become more obvious in regions where
suitable sandfly vectors do not exist. Aggressive interactions
between dogs also have the potential to transmit infection, but
this route has not been proven.
Canine visceral leishmaniosis in dogs and cats is caused by
Leishmania infantum (also referred to as Leishmania chagasi
in Central and South America). L. infantum is endemic in the
Mediterranean basin, the Middle East, and Asia and may have
been introduced to Latin America by early explorers and their
domestic dogs.8 Because leishmaniosis has a long incubation
period and prolonged disease course, travel-related leishmaniosis
in dogs and cats is diagnosed sporadically in regions where sand-
fly transmission does not occur, such as parts of Scandinavia,
Switzerland, Japan, the United Kingdom, and the western United
States (most often in dogs imported from southern Europe).9-13
In highly endemic regions of the Mediterranean and Brazil,
infection rates among dogs vary widely but may reach 80%.14-17
At least 2.5 million dogs are infected in southwestern Europe
alone.14 The disease may be spreading northward in Europe, and
has recently appeared in nontraveled dogs in countries such as
Germany and Hungary.5,18,19 Spread of Leishmania spp. into
new areas can occur when humans bring infected dogs into
regions where competent insect vectors exist, or when sandfly
populations expand as a result of factors such as climate change.
Risk factors for infection in dogs include age at least 2 years,
prolonged exposure to the outdoors, lack of topical insecticide
use, and, in some studies, a short haircoat.20-23 Some authors have
described a bimodal age distribution that peaks at 3 years and at
 CHAPTER 74  Leishmaniosis 715

greater than 8 years of age.24 Sex is not an important risk factor

for infection. Factors that promote proliferation of sandfly popula-
tions, such as poor sanitation and limited garbage collection, as
well as the occurrence of stray dogs around homes, also put dogs at
risk of infection.25 In Europe, German shepherd dogs, boxers, Rott-
weilers, cocker spaniels, and Dobermans may be at increased risk
for leishmaniosis; the Ibizan hound in Spain appears to be resistant
to clinical disease.24,26-29 Concurrent diseases are common in dogs
with leishmaniosis in endemic regions and include transmissible
venereal tumor,30 demodicosis,31 sarcoptic mange,32 and a variety
of other vector-borne diseases such as canine monocytic ehrlichio-
sis, babesiosis, hepatozoonosis, and dirofilariasis.32-36 Concurrent
lymphoma has also been described.32
Uncommonly, dogs worldwide are infected by other spe-
cies of Leishmania, which include Leishmania donovani,
Leishmania tropica, Leishmania braziliensis, Leishmania
peruviana, Leishmania mexicana, Leishmania panamensis,
Leishmania guyanensis, Leishmania amazonensis, and other
as-yet-uncharacterized species.37-45 L. braziliensis, L. guyanensis,
and L. panamensis have been isolated from dogs with Ameri-
can tegumentary leishmaniosis. Outbreaks of American tegu-
mentary leishmaniosis have occurred in humans and dogs.43

Canine Leishmaniosis in North America

Most sporadic cases of visceral leishmaniosis in dogs in North
America are diagnosed in dogs with a travel history to other con-
tinents. Infected dogs continue to be introduced every year as a
result of travel from endemic regions in Europe and Latin America.
In addition, over the past 30 years, visceral disease has been
described in dogs in the United States that have had no apparent
travel history. These have included a basenji dog from a kennel
in Texas and dogs of foxhound breed from Ohio, Oklahoma,
and Colorado.46-49 Involvement of multiple dogs in a foxhound
kennel was reported in Oklahoma,47 and outbreaks in foxhounds
have also been described in New York, Alabama, and Michigan.50
Spread to other dogs has occurred as a result of transplacental
transmission and, in rare cases, transfusion of infected blood.4,6
A competent arthropod vector for Leishmania spp. has not yet
been identified in the United States, and infection has not been
detected in surveys of non-foxhound dogs in the United States.51
Clinical Features
Signs and Their Pathogenesis
The outcome of Leishmania spp. infection is variable. Some dogs
completely eliminate the infection, and a small percentage of dogs
develop severe, life-threatening disease. Some dogs remain persis-
tently but subclinically infected, with the possibility of reactivation
later in life with immunosuppression; these dogs also act as reser-
voir hosts and can infect sandflies.52 In general, dogs that mount
strong T-cell–mediated immune responses eliminate the infection,
whereas those that mount weak cell-mediated immune responses
and marked, nonprotective humoral (IgG) immune responses
develop clinical disease.53 Host genetic factors are important in
determining progression to clinical disease, and several candidate
genes have been identified in dogs that may influence susceptibil-
ity.28,54-56 When clinical disease appears, it occurs after a long
incubation period (up to 7 years) and varies from mild papular or
exfoliative dermatitis to severe disseminated disease due to mas-
sive proliferation of histiocytes, B lymphocytes, and plasma cells
in reticuloendothelial tissues and immune-mediated consequences
of chronic, persistent infection.
FIGURE 74-3  Periocular alopecia and crusting in an 8-year-old male neutered husky
mix with visceral leishmaniosis. The dog was evaluated in northern California but had a
travel history to Spain. Lesions are also present on the muzzle and the pinnae.
Cutaneous lesions most commonly manifest as alopecia,
scaling, and/or ulceration but can be nodular or papular (Figure
74-3). They are often noticed before signs of systemic infection
develop, despite the fact that they occur as a consequence of
parasite dissemination. Many dogs also develop onychogryphosis
(abnormally long or brittle claws).
Systemic signs of visceral leishmaniosis include fever, weight
loss, muscle atrophy, inappetence, and lethargy; oral ulceration;
progressive splenomegaly and lymphadenomegaly; mucosal
pallor due to anemia; and rarely, hepatomegaly (Table 74-1).
Development of autoantibodies and circulating immune com-
plexes is thought to lead to immune-mediated thrombocytope-
nia and/or thrombocytopathia and signs such as epistaxis or
melena, lameness and joint swelling due to immune-mediated
polyarthritis, myositis, uveitis, vasculitis, and glomerulone-
phritis.57-62 Positive antinuclear antibody tests are found in up
to 50% of dogs tested.32,61,63 Many dogs also have positive
Coombs’ tests.32,63
Glomerular injury ultimately culminates in nephrotic syn-
drome and/or failure of tubular function for many dogs, with
polyuria and polydipsia, vomiting, diarrhea, and dehydration.
Renal failure may occur in the absence of other lesions.32 Anemia
may be due to a combination of factors that include blood loss,
inflammation, renal failure, immune-mediated destruction, and
marrow aplasia or hypoplasia. In addition to uveitis, other ocu-
lar lesions include blepharitis, conjunctivitis, panophthalmitis,
and keratoconjunctivitis. Keratoconjunctivitis results from direct
infection of the lacrimal gland by Leishmania.64
Atypical presentations of canine visceral leishmaniosis have
also been reported. Almost any organ can be affected, with
reports of Leishmania prostatitis and infertility65; nodular lesions
716 SECTION 4  Protozoal Diseases

TABLE 74-1
Clinical Signs in Dogs with Visceral Leishmaniosis
Clinical Sign Prevalence (%)
Lymphadenomegaly 65-90
Cutaneous lesions 51-89
Weight loss 25-64
Lethargy 11-60
Pallor 58
Splenomegaly 10-53
Polydipsia 4-40
Abnormal locomotion or polyarthritis 3-38
Pyrexia 4-36
Onychodystrophy 20-31
Diarrhea 30
Vomiting 26
Ocular lesions (keratoconjunctivitis, 7-24
uveitis, panophthalmitis, blepharitis)
Inappetence 17 FIGURE 74-4  Poor body condition of 8-year-old male intact foxhound that was
Polyphagia 15 seropositive and PCR positive for Leishmania infantum. (Courtesy Dr. Christine Petersen,
Iowa State University.)
Epistaxis 6-15
Melena 13
Onychogryphosis may be present. Lameness or reluctance to
Data from Ciaramella P, Oliva G, Luna RD, et al. A retrospective clini- move may be associated with joint swelling, crepitus, and pain.72
cal study of canine leishmaniasis in 150 dogs naturally infected by Leish- Epistaxis may also be noted.
mania infantum. Vet Rec 1997;141:539-543; Slappendel RJ. Canine
leishmaniasis. A review based on 95 cases in The Netherlands. Vet Q
1988;10:1-16; Koutinas AF, Carlotti DN, Koutinas C, et al. Claw his- Diagnosis
topathology and parasitic load in natural cases of canine leishmaniosis
associated with Leishmania infantum. Vet Dermatol 2010;21:572-577. Diagnosis of leishmaniosis may be straightforward, such as
when organisms are seen cytologically in impression smears of
typical skin lesions, or it may require assessment of the results of
of the tongue and other mucosal surfaces66,67; pancreatitis68; gas- a combination of diagnostic assays in light of clinicopathologic
trointestinal tract, myocardial, or pulmonary involvement69-71; findings (Table 74-2). Specific diagnostic assays include cytologic
osteomyelitis72,73; meningitis74; and granuloma formation within or histopathologic examination of affected tissues, serology, cul-
the spinal cord.75 ture of the organism, or the results of PCR assays on affected
tissues or blood. In endemic areas, diagnosis may be challenging
American Tegumentary Leishmaniosis (1) because dogs may be infected with the parasite for long peri-
American tegumentary leishmaniosis in dogs is characterized by ods of time in the absence of clinical signs, so the significance of
development of a nodular skin lesion at the site of a sandfly bite positive test results may be unclear, and (2) because co-infections
that enlarges and eventually may ulcerate. Lesions most often with other vector-borne pathogens may complicate the clinical
occur on regions of the body that have less hair, such as the picture and cause signs that resemble those of leishmaniosis.
nose, perineal region, scrotum, and pinnae.43,76
Laboratory Abnormalities
Physical Examination Findings Complete Blood Count
Physical examination findings in dogs with visceral disease The most common CBC abnormality in dogs with leishmaniosis
include lethargy, muscle atrophy or cachexia, variable and is a mild to moderate, normocytic, normochromic, nonregen-
typically low-grade fever, focal or generalized peripheral erative anemia. Mild thrombocytopenia is present in up to 50%
lymphadenomegaly, and hepatosplenomegaly on abdominal of affected dogs.32,63,77 Total leukocyte, neutrophil, lympho-
palpation (Figure 74-4). Lymphadenomegaly can be severe and cyte, monocyte, and eosinophil counts may be decreased, within
be suggestive of lymphoma. Although not always detected, skin the reference range, or increased. Pancytopenia may be present.
lesions consist of scaling and crusting, hyperkeratosis, alope- Although most dogs are lymphopenic, some have moderate to
cia, erythema, and cutaneous ulceration and may be nonpru- severe lymphocytosis.77
ritic or pruritic. They most often occur on the limbs, pinnae,
muzzle, and periocular regions. Mucocutaneous lesions can Serum Biochemical Tests
also occur. Ocular involvement may be associated with muco- Changes on serum biochemistry analysis in dogs with leish-
purulent ocular discharge, blepharitis, and anterior uveitis. maniosis include hyperglobulinemia, hypoalbuminemia, and
 CHAPTER 74  Leishmaniosis 717

TABLE 74-2
Diagnostic Assays Currently Available for Leishmaniosis in Dogs and Cats
Assay Specimen Type
Cytologic Aspirates of lymph nodes,
examination spleen, or bone marrow;
impression smears of skin
­lesions; rarely blood smears
or buffy-coat preparations
Leishmania culture Aspirates or biopsies of
affected tissues
Antibody serology Serum
(IFA or ELISA)
Real-time PCR Conjunctival swabs, aspirates
assays of affected tissues, lymph
node, bone marrow, whole
blood, skin biopsies
Histopathology Biopsy specimens from
affected tissues (e.g., skin
lesions)
Target
Leishmania infantum
amastigotes within
mononuclear phagocytes
L. infantum
Antibodies to L. infantum
L. infantum DNA
L. infantum amastigotes
Performance
In the absence of experience, organisms
may be confused with fungi such as
Histoplasma capsulatum or Sporothrix
schenckii. Organisms may be few in
number and in some cases are absent.
Sensitive and specific but requires special
techniques and expertise that are not
widely available.
Most infected dogs test positive (sensi-
tivity > 90%), but positive test results
occur in healthy animals, so results
must be interpreted in light of clinical
signs. Sensitivity may be lower in cats.
False positives can occur in dogs
exposed to Trypanosoma spp. (IFA)
and dogs vaccinated with Leishmania
vaccines.
May be useful in dogs with suggestive
lesions in which organisms cannot be
found cytologically. Sensitivity and
specificity depends on assay design.
Quantitative PCR may be useful for
evaluation of treatment efficacy.
Use of immunohistochemistry may
increase sensitivity and can be used to
confirm the presence of Leishmania spp.
in tissues.

IFA, Immunofluorescent antibody.

mild to moderate azotemia.32,63,77 Hyperglobulinemia and
hypoalbuminemia are present in more than 75% of affected
dogs. Serum globulin concentration ranges from mild to
severe (up to 8 g/dL) and reflects a polyclonal gammopa-
thy; monoclonal gammopathy is rare.78 Azotemia has been
reported in 16% to 38% of dogs. Some studies have shown
mild to moderate increases in liver enzyme activities.
Urinalysis
Dogs with leishmaniosis may have proteinuria or isosthenuria,
secondary to glomerular and tubulointerstitial renal function
impairment, respectively. Cylindruria may be detected.
Synovial Fluid Examination
Cytologic examination of synovial fluid from dogs with
Leishmania polyarthritis reveals increased numbers of neu-
trophils and/or lymphocytes.72 In some cases, Leishmania
amastigotes are visible within macrophages in the synovial
fluid.
Bone Marrow Examination
Bone marrow cytologic alterations described in canine leish-
maniosis include erythrophagocytosis, erythroid hypoplasia and
dysplasia, eosinophilic hypoplasia, granulocytic hyperplasia,
and increased numbers of lymphocytes and plasma cells.79,80
Amastigotes may also be visible.
Diagnostic Imaging
Abdominal radiography or ultrasonography in dogs or cats
with visceral leishmaniosis can reveal abdominal lymphadeno-
megaly, splenomegaly, and hepatomegaly. Radiographic lesions
of long bones in dogs with bone involvement consist of soft
periosteal proliferation, increased or decreased intramedullary
opacity, and/or cortical and medullary destruction.63,72 Lesions
tend to be bilateral and symmetrical. Joint radiographs in dogs
with signs of arthritis may reveal erosive changes, or arthritis
may be non-erosive.
Microbiologic Tests
Cytologic Examination
Identification of Leishmania spp. amastigotes in lesions with
cytology is diagnostic for leishmaniosis (Figure 74-5). Organ-
ism densities are particularly high in skin lesions, bone marrow,
and the spleen, but specimens collected from other tissues, such
as lymph node and hepatic aspirates, may also contain organ-
isms. Organisms are not found in some lesions, and inexperi-
enced microscopists may fail to identify organisms when present
or confuse them with other parasites, such as Trypanosoma,
718 SECTION 4  Protozoal Diseases
Histoplasma, or Sporothrix schenckii. Rarely, organisms are
seen in circulating white blood cells.81
Serologic Diagnosis
Serologic assays based on immunofluorescent antibody (IFA),
ELISA, Western immunoblot, and agglutination techniques
have all been used for serodiagnosis of canine leishmaniosis,
or for screening apparently healthy dogs in endemic areas for
evidence of infection. IFA is commonly used by veterinary
practitioners in Europe82 and has a diagnostic sensitivity that
ranges from 90% to 100% and a specificity of 80% to 100%
in dogs that have not received a Leishmania vaccine. Dogs
with Trypanosoma spp. infections may have false-positive test
results due to serologic cross-reactivity between Trypanosoma
and Leishmania antigens. ELISA assays based on recombinant
antigens often have higher specificity and are technically less
difficult to perform than IFA.42,83 A rapid lateral flow assay is
available commercially (SNAP Canine Leishmania Antibody
Test Kit, IDEXX Laboratories) that had an overall sensitivity
of 95% and a specificity of 91% in one study of 400 dogs from
Brazil.83 Other immunochromatographic assays are available
that vary in their sensitivity.
Negative serologic test results may reflect delayed seroconver-
sion in some animals, which can take several months to occur.
However, many dogs with clinical signs have already serocon-
verted because of the long incubation period. Importantly, in
endemic areas, positive serologic test results do not imply that
disease is due to Leishmania spp. infection, because subclini-
cal infections are widespread. Thus, for diagnosis of clinical
leishmaniosis, results must be interpreted in conjunction with
clinical findings and cytologic and histopathologic evaluation
of affected tissues. Because seroconversion occurs in dogs that
have received the Leishmania vaccine, current serologic assays
cannot be used to efficiently differentiate between infected and
vaccinated animals.84
FIGURE 74-5  Bone marrow aspirate cytology findings in a dog with visceral leish-
maniosis. Numerous Leishmania amastigotes can be seen within a histiocyte. (Courtesy
Dr. Sheri Ross, University of California, Davis – San Diego.)
Molecular Diagnosis Using the Polymerase Chain Reaction
Several different conventional and real-time PCR assays have
been developed and evaluated for detection of Leishmania spp.
in dogs and cats. Real-time PCR assays that detect Leishma-
nia spp. are now offered on a commercial basis by some veteri-
nary diagnostic laboratories worldwide. Suitable specimens for
testing include splenic, hepatic, lymph node, or bone marrow
aspirates; whole blood; skin biopsies; conjunctival swabs; and
even oral swabs. Conjunctival swabs; aspirates of spleen, bone
marrow, or lymph nodes; and skin biopsies are most likely to
yield positive results.85,86 Positive PCR assay results in endemic
areas do not confirm that disease is caused by Leishmania spp.,
because dogs without clinical signs can test positive.87 However,
in one study, clinical disease subsequently appeared in 71% of
ELISA-positive dogs that lacked clinical signs but had medium
to high parasite loads (more than 10 parasites/mL of blood) as
determined with a quantitative PCR assay.86 PCR assays may
also be useful in order to monitor the efficacy of treatment.
Culture
Leishmania spp. can be isolated in culture from specimens
such as lymph nodes, skin biopsies, spleen, or bone marrow,
although special media and expertise are required, and incuba-
tion times of up to 30 days may be needed.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with visceral leishmaniosis
include cachexia; generalized enlargement of the spleen, lymph
nodes, and sometimes the liver; focal pale nodular lesions in a
variety of organs; bone and joint lesions; and gastrointestinal
ulcerations.
Histopathologic Findings
Histopathology of most affected tissues in dogs with leish-
maniosis typically reveals granulomatous to pyogranuloma-
tous and lymphoplasmacytic inflammation, with variable
numbers of intrahistiocytic amastigotes (Figure 74-6). Skin
lesions are accompanied by orthokeratotic to parakeratotic
FIGURE 74-6  Histopathology of the spleen in a dog with visceral leishmaniosis. Mul-
tiple amastigotes (which have a granular appearance, arrows) are present within macro-
phages. H&E stain; bar = 20 µm. (Courtesy Dr. Christine Petersen, Iowa State University.)
 CHAPTER 74  Leishmaniosis 719

hyperkeratosis, acanthosis, and ulceration. Claw histopathol-
ogy in dogs with onychogryphosis reveals lichenoid mono-
nuclear cell infiltration, with or without basal keratinocyte
vacuolation and dermo-epidermal clefting; organisms are
generally not visible.88 Renal lesions consist of membranop-
roliferative glomerulonephritis, lymphoplasmacytic tubuloint-
erstitial nephritis, and rarely amyloidosis (Figure 74-7).46,89-91
Immunohistochemistry can be used to unambiguously detect
Leishmania amastigotes in tissue sections. Immunohistochem-
istry may permit detection of Leishmania spp. when organisms
are not seen with routine histopathology.70,92 In situ hybrid-
ization has also been used to identify Leishmania amastigotes
in paraffin-embedded specimens.92
Treatment and Prognosis
Antiprotozoal Therapy
Drugs with activity against Leishmania spp. include the pen-
tavalent antimonials meglumine antimoniate (Glucantime) and
FIGURE 74-7  Histopathology of the kidney in a dog with visceral leishmaniosis.
There is marked thickening of glomerular capillary loops (arrows) indicative of glomerulo-
nephritis. H&E stain. Bar = 50 µm. (Courtesy Dr. Christine Petersen, Iowa State University.)
sodium stibogluconate; allopurinol; amphotericin B; and miltefos-
ine (Table 74-3). Ketoconazole has also been used. Unfortunately,
regardless of the treatment used, complete parasitologic cure is
rare, and relapses are frequent.93,94 In endemic areas, reinfec-
tions occur and contribute to apparent treatment failures. Staging
systems and consensus treatment guidelines have been published
(Table 74-4).29,95
Pentavalent antimonials are thought to inhibit protozoal
enzymes and damage protozoal DNA, whereas allopuri-
nol interferes with protein synthesis by Leishmania spp. (see
Chapter 10 for more information on these drugs). In general,
a combination of meglumine antimoniate (100 mg/kg SC
q24h for 4 weeks) and allopurinol (10 mg/kg PO q12h until
clinical signs have resolved and quantitative serology becomes
negative) is the treatment of choice for dogs with leishmani-
osis.96 Clinical improvement generally occurs within 1 to 2
months of treatment initiation if no overt renal disease is pres-
ent. Complete or near-complete clinical remission rates of
65% to 100% have been reported with combination therapy,
with relapse rates of around 10%.93,97 Dogs that relapse can
be retreated with one or more cycles of meglumine. Relapses
can occur years after treatment is initiated, even in the face
of allopurinol treatment. Rarely, xanthine urolithiasis com-
plicates long-term allopurinol treatment.97 A liposomal form
of meglumine antimoniate (LMA) (6.5 mg/kg of stiboglu-
conate every 4 days), when used together with allopurinol
(20 mg/kg PO q24h) and administered for 140 days, signifi-
cantly reduced clinical signs and parasite load and blocked
transmission of the parasite to sandflies, but was associated
with apparent cure in only 50% of treated dogs.98 Unfortu-
nately, resistance to antimonials has emerged among some
Leishmania isolates in highly endemic areas such as southern
Europe. Antimonials are also not readily available for treat-
ment of dogs in the United States.
Miltefosine activates proteases in Leishmania spp. and
causes apoptotic death of the parasite (see Chapter 10). Milt-
efosine can be used as an alternative to meglumine antimoni-
ate in conjunction with allopurinol for 1 month, after which
monotherapy with allopurinol is continued.99 Unfortunately
this combination protocol does not result in parasitologic cure,
and relapse is common. Even a second cycle of miltefosine treat-
ment failed to eliminate the parasite in one study.99

TABLE 74-3
Suggested Drug Doses for Treatment of Canine Visceral Leishmaniosis
Drug Dose (mg/kg) Route Interval (hours) Duration and Comments
Allopurinol 10 PO 12 At least 6 to 12 months or until both clinical signs
disappear and quantitative serology becomes
negative
Meglumine 75-100 SC 24 30 days. Use with allopurinol. Meglumine antimoniate
antimoniate 50 12 is the first-line treatment where available.
Miltefosine 2 PO 24 28 days. Use with allopurinol where miltefosine is
available.
Lipid-complexed 3 IV Every 48 hours on Dilute to 1 mg/mL in D5W and administer over
amphotericin B Monday, Wednesday, 1 to 2 hours until a cumulative dose of 21 mg/kg
and Friday is reached. Monitor kidney values (see Chapter
9). Optimal dosing schedule for dogs is unknown.
720 SECTION 4  Protozoal Diseases

TABLE 74-4
Clinical Staging of Canine Visceral Leishmaniosis Based on Serologic Status, Clinical Signs, Laboratory Findings,
Type of Treatment, and Prognosis for Each Stage
Clinical Laboratory
Stage Antibody Titer Clinical Signs Findings Therapy Prognosis
Stage I Negative or low Mild signs such as Usually none, creatinine None, allopurinol Good
positive (<3- to peripheral lymphade- <1.4 mg/dL, UPC <0.5 monotherapy,
4-fold increase above nopathy or papular or combination
the laboratory cutoff) dermatitis therapy
Stage II Positive (low or high) Diffuse or symmetrical Anemia, hyperglobulinemia, Combination Good to guarded
cutaneous lesions, and/or hypoalbuminemia therapy
ulcerations, anorexia, present.
weight loss, fever, Substage a: nonazotemic,
and/or epistaxis nonproteinuric
Substage b: nonazotemic,
UPC 0.5-1
Stage III Medium to high Stage II abnormalities Stage II abnormalities Combination Guarded to poor
plus signs of immune- plus IRIS* stage I with therapy
complex disease UPC >1, or IRIS stage II
(vasculitis, arthritis,
glomerulonephritis)
Stage IV Medium to high Stage III abnormali- Stage II abnormalities Consider mono- Poor
ties plus nephrotic plus IRIS stage III or IV; therapy with al-
syndrome, pulmonary marked proteinuria lopurinol. Follow
thromboembolism, (UPC >5) IRIS guidelines
or end-stage renal for treatment of
disease chronic kidney
disease.

Data from Solano-Gallego L, Koutinas A, Miro G, et al. Directions for the diagnosis, clinical staging, treatment and prevention of canine leishmaniosis.
Vet Parasitol 2009;165:1-18.
*International Renal Interest Society, http://www.iris-kidney.com/guidelines/en

Lipid-complexed amphotericin B is the treatment of choice
for human visceral leishmaniasis. Limited reports of the use
of both deoxycholate and lipid-complexed forms of ampho-
tericin B to treat dogs with leishmaniosis exist.100,101 The
need for intravenous administration of amphotericin B and
its nephrotoxicity has limited its use for treatment of dis-
ease in dogs. In one study, use of liposomal amphotericin B
(Ambisome) to treat canine leishmaniosis was associated
with rapid resolution of clinical signs, but cytologic evidence
of parasites persisted in lymph node aspirates.100 However,
treatment was administered for less than 2 weeks (and for
some groups of dogs in the study, only 3 days). The FDA-
approved regimen for liposomal amphotericin B treatment
of immunocompetent human patients consists of seven infu-
sions over 21 days (days 1 through 5, 10, and 21), for a total
dose of 21 mg/kg.102
Because of the likelihood of relapse in dogs after treatment, the
World Health Organization does not advocate treatment of pet
dogs with anti-Leishmania chemotherapy protocols used to treat
humans and instead suggests monotherapy with allopurinol.103
Supportive Care
Dogs with renal failure secondary to Leishmania infection may
require supplemental fluid therapy and medications that address
complications of uremia and/or protein-losing nephropathy,
such as antacids, antiemetics, phosphate binders, low-protein
diets, and antihypertensive drugs.
Immunity and Vaccination
Although immunity to leishmaniosis is complex and not well
understood, it is clear that it is strongly dependent on the develop-
ment of a robust Th1-cell–mediated immune response. Production
of cytokines such as IFN-γ and TNF-α activates macrophages to
destroy amastigotes, which occurs through nitric oxide–­mediated
mechanisms. Dogs that resist leishmaniosis exhibit strong delayed
type hypersensitivity responses to leishmanial antigen when it is
administered intradermally. A balance between Th1 and Th2
immune responses appears to be important in determining the
clinical outcome of infection.
Several vaccines against leishmaniosis have been evaluated in
dogs under field conditions. One of these vaccines, Leishmune,
became licensed for prevention of leishmaniasis in dogs in Brazil
in 2011.53 Leishmune is composed of a promastigote antigen
(fucose-mannose ligand) and the adjuvant saponin.104,105 The
vaccine has not been approved in Europe because of adverse
effects induced by the saponin adjuvant. Efficacy in initial
field trials was approximately 80% and has correlated with a

reduction in human leishmaniasis in the area.53 The vaccine is
also licensed in Brazil for immunotherapy, and reduces clinical
signs in infected dogs,106 but when used without antiprotozoal
drugs did not reduce 4.5-year mortality when compared with
infected control dogs.107 Another vaccine (CaniLeish, Virbac)
composed of purified excreted-secreted proteins of Leishmania
infantum (LiESP) is licensed in Europe for prevention of canine
leishmaniosis. This vaccine stimulates a Th1-dominated anti–L.
infantum response within 3 weeks of administration.108
Prevention
In addition to vaccination, use of ectoparasiticides such as
­deltamethrin-impregnated dog collars or spot-on repellants
that contain permethrin can prevent canine leishmaniosis.111-114
Indoor housing or use of fine-mesh netting around dogs and
humans when sandflies feed at night (from dusk to dawn) is also
recommended. Because of transplacental transmission, neuter-
ing may also reduce the spread of Leishmania.6 Potential blood
donors should be screened with serology and PCR assays and
should only be used if both assays are negative.29
A dopamine-based oral suspension Leisguard is available in
some parts of Europe for prevention of leishmaniosis in dogs
that travel to regions where the disease is endemic. This con-
tains the dopamine D2 receptor antagonist domperidone, which
activates phagocytic cells and enhances intracellular killing
of the parasite.109 A prospective, randomized controlled trial
showed that Leisguard prevented transmission of leishmaniosis
to naïve dogs in a kennel.115 The drug was administered q24h
for two 30-day periods at the estimated beginning and end of
the vector activity period. Leisguard may also hasten recovery
when used for treatment of affected dogs in combination with
meglumine antimoniate and allopurinol,110 but additional stud-
ies are required.
Public Health Aspects
An estimated 12 million people worldwide have leishmani-
asis, and at least 350 million people are estimated to be at risk.
Approximately 0.2 to 0.4 million new cases of VL and 0.7 to
1.2 million cases of CL are believed to occur in humans annu-
ally, and it has been estimated that 20,000 to 40,000 leishmani-
asis deaths occur per year.116
VL in humans is also known as kala-azar. It is caused mostly
by L. donovani and L. infantum (referred to as L. chagasi in Cen-
tral and South America), but also by L. tropica. More than
90% of VL cases globally occur in India, Bangladesh, Sudan,
South Sudan, Brazil, and Ethiopia. Many cases are due to L.
donovani, for which humans are the primary reservoir.116
L. infantum causes VL in central Asia, China, the Mediter-
ranean basin, the Middle East, and Latin America. Most dis-
ease associated with L. infantum infection occurs in children
(infantile leishmaniasis) and immunocompromised adults,
such as those with AIDS. L. infantum is also responsible for
some CL in humans. Dogs are considered the primary reser-
voirs for L. infantum, and control of disease in dogs has been
CHAPTER 74  Leishmaniosis 721
associated with reduction in human cases in the area. Nev-
ertheless, other canine species, such as foxes, may also play
a role as reservoirs.
Human CL occurs in the Americas, the Mediterranean
basin, and western Asia from the Middle East to Central Asia,
and especially in Afghanistan, Algeria, Colombia, Brazil, Iran,
Syria, Ethiopia, North Sudan, Costa Rica, and Peru. In Africa
and western Asia, CL is caused by L. major, L. tropica, and
L. aethiopica, whereas in the Americas, the most common
causes are L. mexicana and L. braziliensis. Mucocutaneous
disease occurs primarily in South America and is caused by
L. braziliensis, for which rodents and other small mammals are
the primary reservoirs.117
Recommendations of the World Health organization for
the control of leishmaniasis in humans consist of treatment of
human disease; culling of wild and feral seropositive dogs; and
insecticidal treatment of human homes, such as with pyrethroid
sprays.103 The effectiveness of the culling of seropositive or
infected dogs is controversial.42,118 At present this is only under-
taken systematically in Brazil. Concerns raised include the ethics
of destruction of pet dogs, delays between detection of seroposi-
tive dogs and culling, and the persistence of other subclinically or
clinically infected canine and wildlife reservoirs. Vaccination of
dogs may be more effective than the culling of seropositive dogs
and could overcome the ethical concerns that relate to culling.53
FELINE LEISHMANIOSIS
Cats are relatively resistant to leishmaniosis, but cases have
been described from southern Europe, the Middle East,
Latin America, and Texas.119,120 The cat from Texas was
infected with L. mexicana. L. infantum and a variety of other
Leishmania species such as L. braziliensis and L. venezu-
elensis have also been isolated from cats. Serologic studies
of cats in endemic areas have generally revealed seropreva-
lences of less than 10%, although 34 (36%) of 95 stray cats
from the Yucatán peninsula in Mexico had antibodies to
L. infantum, L. braziliensis, or L. mexicana.119 Disease in cats
may be associated with concurrent FIV infection.121,122 The
extent to which cats are a reservoir for infection of humans
is unclear.
Clinical signs of leishmaniosis in cats include cutaneous
lesions, weight loss, lymphadenopathy, oral ulceration, muco-
purulent ocular and nasal discharge, keratitis, altered mentation,
diarrhea, and/or tachypnea. Cutaneous lesions consist of nodules,
scaling and crusting, erythema, ulceration, and alopecia. Cutane-
ous lesions can occur anywhere and may be localized or general-
ized, but are often on the pinnae, bridge of the nose, limbs, or
dorsal trunk. In one study, 50% of cats with skin lesions had
evidence of visceral spread.121
Diagnosis of leishmaniosis in cats is similar to that in dogs,
although cats often have low antibody titers.121 Limited infor-
mation exists in regard to treatment of feline leishmaniosis.
Treatment with allopurinol monotherapy may result in clinical
improvement or disease resolution in some cats.123 Surgery may
be curative for localized cutaneous lesions.12
722 SECTION 4  Protozoal Diseases
CASE EXAMPLE
Signalment: “Fausto,” an 8-year-old male neutered husky mix
from northern California
History: Fausto was referred to the University of California, Davis,
Veterinary Medical Teaching Hospital (VMTH) for evaluation
of suspected autoimmune disease. The dog had a 6-month
history of progressive skin lesions that began as crusting and
ulceration around the elbow and stifle joints. This was followed
by development of similar lesions above both eyes and on
the dog’s ears and dorsal muzzle. Three months after the skin
lesions appeared, Fausto became stiff and lethargic and was
lame and reluctant to walk. At that time, Fausto was taken to
a local veterinary clinic where an injection of triamcinolone
acetonide (Vetalog) was administered, and he was prescribed
carprofen and trimethoprim-sulfamethoxazole. There was
no improvement in the clinical signs. The day before he
was brought to the VMTH, the owner had taken Fausto to a
local emergency clinic because he was reluctant to move.
Blood work and arthrocentesis (left and right tarsal joints)
were performed. The blood work showed a normocytic,
normochromic nonregenerative anemia (HCT 31%); mild
hypoalbuminemia (2.4 g/dL); and moderate hyperglobulinemia
(7.7 g/dL). Cytologic examination of the synovial fluid revealed
large numbers of nondegenerate neutrophils, which was
considered consistent with immune-mediated polyarthritis.
Cephalexin and carprofen were prescribed, and the dog was
referred for further evaluation.
The owner had not noticed any coughing, sneezing, vomit-
ing, diarrhea, or changes in thirst or urination. Fausto was
fed 3 cups of a commercial dry chicken and rice–based
dog food once a day, and his appetite was good. He
was fully vaccinated for rabies, canine distemper virus,
canine adenovirus, and canine parvovirus. The dog did
not receive heartworm prophylaxis medication but was
treated with fipronil once a month for flea control. There
was one other healthy dog in the household. The owner
and his dogs had moved to California from Barcelona,
Spain, a few weeks earlier, around the time that the skin
lesions appeared. In Spain, Fausto had worn a collar to
prevent insect bites.
Physical Examination:
Body Weight: 34.4 kg.
General: Quiet, alert, and responsive, but reluctant to ambulate.
T = 102.7°F (39.3°C), HR = 132 beats/min, panting but eupneic,
mucous membranes moist and pink.
Integument: Multiple focal regions of crusting, erythema,
hyperpigmentation, thickening, and sometimes ulceration
were present over the elbows and stifles, prepuce, ventral
aspect of the mandible, above both eyes, and on both pinnae.
Moderate pruritis was present.
Musculoskeletal: BCS 5/9. The dog was able to walk but had
a shifting lameness. Pain was detected on extension of the
joints, but there was no palpable effusion present.
Abdominal Palpation: Splenomegaly was detected.
Lymph Nodes: Superficial cervical and popliteal lymph nodes
were all enlarged (approximately 3 cm in diameter).
All Other Systems: No significant abnormalities were detected.
Laboratory Findings:
CBC:
HCT 32.4% (40%-55%)
MCV 62.7 fL (65-75 fL)
MCHC 35.2 g/dL (33-36 g/dL)
Reticulocyte count 54,500 cells/µL
WBC 9300 cells/µL (6000-13,000 cells/µL)
Neutrophils 7533 cells/µL (3000-10,500 cells/µL)
Lymphocytes 930 cells/µL (1000-4000 cells/µL)
Monocytes 651 cells/µL (150-1200 cells/µL)
Eosinophils 186 cells/µL (0-1500 cells/µL)
Platelets 338,000 platelets/µL (150,000-400,000 platelets/µL).
Serum Chemistry Profile:
Sodium 140 mmol/L (145-154 mmol/L)
Potassium 4.4 mmol/L (4.1-5.3 mmol/L)
Chloride 107 mmol/L (105-116 mmol/L)
Bicarbonate 15 mmol/L (16-26 mmol/L)
Phosphorus 3.8 mg/dL (3.0-6.2 mg/dL)
Calcium 10.4 mg/dL (9.9-11.4 mg/dL)
BUN 16 mg/dL (8-31 mg/dL)
Creatinine 0.9 mg/dL (0.3-1.2 mg/dL)
Glucose 109 mg/dL (70-118 mg/dL)
Total protein 10.7 g/dL (5.4-7.4 g/dL)
Albumin 2.1 g/dL (2.9-4.2 g/dL)
Globulin 8.6 g/dL (2.3-4.4 g/dL)
ALT 12 U/L (19-67 U/L)
AST 39 U/L (21-54 U/L)
ALP 43 U/L (15-127 U/L)
GGT 3 U/L (0-6 U/L)
Cholesterol 191 mg/dL (135-345 mg/dL)
Total bilirubin 0.1 mg/dL (0-0.4 mg/dL).
Serum Protein Electrophoresis: There was a marked increase
in gamma-globulin with mild hypoalbuminemia. The
gammopathy was polyclonal.
Urinalysis (Cystocentesis): SGr 1.037; pH 7.0, 4+ protein, 1+
bilirubin, 1+ hemoprotein, no glucose or ketones, rare WBC/
HPF, 1-3 RBC/HPF, rare hyaline casts, few lipid droplets.
Urine Protein-to-Creatinine Ratio: 2.99 (normal, <0.5).
Microbiologic Testing: IFA serology for antibodies for
Ehrlichia spp., Anaplasma spp., Rickettsia spp., Bartonella
clarridgeiae, Bartonella vinsonii subsp. berkhoffii, and
Bartonella henselae: Negative.
Western immunoblot for antibodies to Borrelia burgdorferi:
Negative.
Knott’s and ELISA antigen test for Dirofilaria immitis infection:
Negative.
Aerobic bacterial, anaerobic bacterial, and Mycoplasma
culture of synovial fluid: Negative.
Aspiration cytology (right and left superficial cervical and popli-
teal lymph nodes): A heterogeneous population of lympho-
cytes was present, which consisted primarily of small mature
lymphocytes and slightly increased numbers of intermedi-
ate-sized lymphocytes and lymphoblasts. Large numbers of
plasma cells were noted. A few macrophages, nondegener-
ate neutrophils, and eosinophils were scattered throughout
the specimen.
Bone marrow aspiration cytology: Many unit particles were pres-
ent. Unit particles were normocellular, and a small amount
of storage iron was noted. There were normal numbers of

megakaryocytes. The myeloid-to-erythroid ratio was mildly
increased; both series were synchronous and complete. In-
creased numbers of plasma cells were noted throughout
the specimen, with few small lymphocytes. Macrophages
occasionally contained intracytoplasmic organisms consis-
tent with Leishmania spp. amastigotes. Interpretation: Mild
erythroid hypoplasia, Leishmania spp., and mild plasma cell
hyperplasia.
Skin biopsy: There was a thick band of inflammatory cells that
extended from the superficial to the mid-dermis. The in-
flammatory infiltrate was composed of dense sheets of
large foamy macrophages admixed with smaller numbers of
lymphocytes and plasma cells and few neutrophils. Many of
the large macrophages contained multiple, round to oval,
intracytoplasmic basophilic organisms that were 1 to 2 µm
in diameter. The epidermis was hyperplastic and hyperkera-
totic and was covered by exudate admixed with keratin, de-
generate neutrophils, and cellular debris.
Diagnosis: Canine visceral leishmaniosis.
Treatment and Outcome: Fausto was initially treated
with a combination of allopurinol (10 mg/kg PO q8h),
ketoconazole (9 mg/kg PO q12h), cephalexin (30 mg/kg PO
q12h for 4 weeks, for secondary bacterial pyoderma), and
carprofen (4.4 mg/kg PO q24h). At a recheck examination 2
weeks later, the dog’s owner reported that 1 or 2 days after
treatment was started, Fausto became intensely pruritic.
Fausto’s appetite also decreased and he developed increased
thirst and urination, but the lameness resolved. Physical
examination showed severe crusting and ulceration of
the face, with an associated foul odor. This extended from
the dorsal nasal planum to the temporal and supraocular
regions, as well as the inner aspect and tips of the pinnae.
The lesions on the limbs were static or slightly improved from
the last visit. A CBC was unchanged, and a serum chemistry
profile showed worsened hyperglobulinemia (9.3 g/dL) and
SUGGESTED READINGS
Maroli M, Gradoni L, Oliva G, et al. Guidelines for prevention of leish-
maniasis in dogs. J Am Vet Med Assoc. 2010;236:1200-1206.
Oliva G, Roura X, Crotti A, et al. Guidelines for treatment of leishmaniasis
in dogs. J Am Vet Med Assoc. 2010;236:1192-1198.
Paltrinieri S, Solano-Gallego L, Fondati A, et al. Guidelines for diagnosis
and clinical classification of leishmaniasis in dogs. J Am Vet Med Assoc.
2010;236:1184-1191.
Solano-Gallego L, Koutinas A, Miro G, et al. Directions for the diagno-
sis, clinical staging, treatment and prevention of canine leishmaniosis.
Vet Parasitol. 2009;165:1-18.
Solano-Gallego L, Miro G, Koutinas A, et  al. LeishVet guidelines for
the practical management of canine leishmaniosis. Parasit Vectors.
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CHAPTER 74  Leishmaniosis 723
hypoalbuminemia (1.9 g/dL). A drug reaction was suspected,
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80. Tropia de Abreu R, Carvalho MG, Carneiro CM, et al. Influence
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91. Zatelli A, Borgarelli M, Santilli R, et al. Glomerular lesions in dogs
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93. Noli C, Auxilia ST. Treatment of canine Old World visceral leish-
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94. Andrade HM, Toledo VP, Pinheiro MB, et  al. Evaluation of
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L. infantum (= L. chagasi) in Brazil. Vet Parasitol. 2011;181:83-90.
95. Oliva G, Roura X, Crotti A, et  al. Guidelines for treatment of
leishmaniasis in dogs. J Am Vet Med Assoc. 2010;236:1192-1198.
96. Paradies P, Sasanelli M, Amato ME, et  al. Monitoring the
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97. Torres M, Bardagi M, Roura X, et al. Long term follow-up of dogs
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98. da Silva SM, Amorim IF, Ribeiro RR, et al. Efficacy of combined
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99. Manna L, Vitale F, Reale S, et al. Study of efficacy of miltefosine and
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100. Oliva G, Gradoni L, Ciaramella P, et  al. Activity of liposomal
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mania infantum. J Antimicrob Chemother. 1995;36:1013-1019.
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104. Parra LE, Borja-Cabrera GP, Santos FN, et al. Safety trial using
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105. Nogueira FS, Moreira MA, Borja-Cabrera GP, et  al. Leishmune
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CHAPTER 75
Babesiosis
Adam J. Birkenheuer
Overview of Babesiosis in Dogs and Cats
First Described: Early descriptions of intraerythrocytic para-
sites in dogs with signs of babesiosis were made in Africa
in 1893 (Hutcheon).1 The first documented case of canine
babesiosis in the United States was in 1934 (Eaton).2
Cause: Babesia canis, Babesia gibsoni, Babesia conradae (dogs);
Babesia felis, Babesia cati (cats), several other unnamed
species (phylum Apicomplexa)
Affected Hosts: Domestic dogs and cats
Geographic Distribution: Worldwide, but different Babesia
species vary in their geographic distribution
Mode of Transmission: A variety of tick vector species; fight-
ing or biting interactions; vertical transmission; blood
transfusion
Major Clinical Signs: Lethargy, pallor, splenomegaly. Dogs
with severe babesiosis caused by B. canis rossi may show
neurologic signs, anuria, tachypnea, and poor perfusion
parameters.
Differential Diagnoses: Hemoplasmosis, primary immune-
mediated hemolytic anemia and/or thrombocytopenia,
other causes of hemolysis such as oxidative toxins or
microangiopathic hemolysis (e.g., with splenic hemangio-
sarcoma). Canine monocytic ehrlichiosis, Rocky Mountain
Spotted Fever, and bacterial endocarditis should also be
considered.
Human Health Significance: Babesia species that infect dogs
and cats are not known to infect humans.
Etiology and Epidemiology
Babesiosis, caused by infection with organisms from the genus
Babesia, is characterized by hemolytic anemia, fever, and sple-
nomegaly. Babesia infections can also be subclinical or cause
severe life-threatening illness. Babesia spp. are intraerythrocytic
protozoan parasites of the phylum Apicomplexa. Altogether,
more than 100 species of Babesia have been described, and
with the advent of molecular techniques such as PCR assays,
new species and genotypes are identified each year. Histori-
cally, species have been named and identified based on the ver-
tebrate host and the size of the parasite (large or small Babesia
species). Large Babesia spp. are 3 to 7 µm in length, whereas
small ­Babesia spp. are 1 to 3 µm in length. In most parts of
the world, tick vectors are the most important means of trans-
mission of Babesia species. However, for some Babesia species,
such as Babesia gibsoni in North America and Europe, direct
transmission between dogs, through fighting (and exchange of
blood), or congenital transplacental transmission, is believed to
be the most common route of transmission when competent tick
vectors are absent.3-5
When ticks are involved in transmission, sporozoites are
released from the tick salivary glands as the tick feeds and they
enter the bloodstream of the vertebrate host (Figure 75-1). They
then attach to, and are endocytosed by, erythrocytes. Within
erythrocytes, they undergo asexual reproduction (merogony),
and the daughter cells infect new erythrocytes. A naïve tick then
ingests infected erythrocytes. It is unclear whether transformation
from merozoite to gametocyte begins in the vertebrate host or
in the tick. In the tick midgut, the sexual phase of reproduction
occurs when the gametocytes fuse to form a zygote. The zygote
invades epithelial cells of the tick gut. The resultant forms, ookine-
tes, leave the epithelial cell and invade either the salivary gland or
the ovary, where they participate in transstadial and transovarial
transmission, respectively. Prior to transmission, another asexual
form of reproduction occurs in the salivary gland, sporogony.
Babesiosis in Dogs
Canine babesiosis is a disease of worldwide importance. Ini-
tially, two species of Babesia were recognized in dogs (Babesia
canis and B. gibsoni); however, now at least nine genetically
distinct canine piroplasms have been described. Babesia species
that infect dogs vary in their geographic distribution, which in
turn has been subject to change due to movement of infected
animals, movement of tick vectors, and reclassification with
improved diagnostic techniques. As new species have been
recognized, it has become more important for the clinician to
understand the differences in diagnosis, prognosis, and treat-
ment for infection with each species.
Babesia canis
Babesia canis is the most common large Babesia species and
has three distinct subspecies: B. canis vogeli, B. canis canis, and
B. canis rossi. It has been proposed that these are in fact three
distinct species (B. vogeli, B. canis, and B. rossi).6 B. canis vogeli
is transmitted by the brown dog tick (Rhipicephalus sanguin-
eus). Infection is most commonly diagnosed in warm, humid
regions of the world, and the disease occurs throughout the year
in endemic regions. B. canis vogeli can be found in Africa, Asia,
Australia, Europe, and the Americas. In the United States, dis-
ease is most often diagnosed in the southern regions. B. canis
vogeli reported seroprevalence in dogs has ranged from 3.8% to
59%.7 The prevalence of seroreactivity is higher in adult dogs
than in dogs younger than 1 year.8 In an antibody serosurvey
of dogs in Florida, 46% of 393 greyhounds were seroreactive.7
The prevalence of seroreactive dogs within these kennels ranged
from 17% to 100%; the lower prevalence was noted in kennels
727
728 SECTION 4  Protozoal Diseases
Tick ovary Transform into
gametes which
Zygote fuse to form
Ookinete zygotes
migration
Tick
gut
Transstadial
and
transovarial
transmission
Rhipicephalus
sanguineus
Ingested
by tick
Tick salivary vector
gland
Sporozoites
Sporogony released into
tick saliva
FIGURE 75-1  Life cycle of Babesia canis.
with more intensive tick control. None of 50 non-greyhound,
individually housed adult pet dogs within the same geographic
area were seroreactive, which implicated environment and breed
susceptibility as possible risk factors in endemic areas.7 Trans-
placental transmission of B. canis vogeli infections is strongly
suspected but unproven in an experimental setting.9
B. canis canis is found in Europe and Africa. It is trans-
mitted by the ornate cow tick, Dermacentor reticulatus, but
R. sanguineus may also be a vector.10 The incidence of infec-
tion is highest in the fall and spring. Higher prevalence rates
are most commonly found in rural or suburban areas that are
adjacent to prairies or woodlands that provide suitable habi-
tat for D. reticulatus.11 B. canis canis infection is most often
diagnosed in France. Infections are being reported more fre-
quently from other European countries such as Croatia, Poland,
and Germany, possibly due to changes in the distribution of
D. ­reticulatus.12 Outbreaks of disease due to B. canis canis infec-
tion in Polish sled dogs share many similarities with those due
to B. canis vogeli infection in North American greyhounds.13
B. canis rossi is transmitted by the yellow dog tick, Haema-
physalis elliptica (formerly Haemaphysalis leachi).14 Reports of
B. canis rossi infection have been limited to Africa, with the vast
majority of reports coming from South Africa, where more than
10% of the dogs evaluated in some veterinary hospitals may
be affected.15,16 The incidence of infection is highest during the
summer months. Breed predispositions for infection have not
been well studied, but the traditional fighting breeds (­American
pit bull terriers, Staffordshire bull terriers, and bull terriers)
are more likely to die when they are diagnosed with severe
babesiosis.17
Unnamed Large Babesia Species
A large Babesia sp. has been isolated from dogs in North
­America that had been splenectomized or were undergoing
chemotherapy for cancer. Infected dogs have been identified in
North Carolina, New Jersey, New York, and Texas.18-20 A tick
vector has not been identified. A different novel large Babesia
Merozoites
released
Merogony
(binary fission)
Ring form
Sporozoites
Tick penetrate
feeds
dog RBC
on dog
sp. was reported in a dog from Great Britain that never traveled
outside of that country.21 The dog died as a result of infection.
Babesia gibsoni
Infections with B. gibsoni occur throughout the world, and the
insidious nature of this infection has allowed the inadvertent
transport of infected dogs from Asia to other areas. B. gibsoni
can be found in Africa, Asia, Australia, North and South Amer-
ica, and Europe. Depending on the availability of suitable vec-
tors, two distinct epidemiologic scenarios exist for B. gibsoni:
tick transmission and direct transmission between dogs.22,23
B. gibsoni is transmitted primarily by the tick vectors Hae-
maphysalis bispinosa and Haemaphysalis longicornis, and
possibly by R. sanguineus.24-28 In its original endemic area of
Asia, the geographic range of B. gibsoni correlates with that of
H. bispinosa, and tick infestation is a risk factor for infec-
tion in nonfighting breeds. In the United States where compe-
tent tick vectors are not endemic, B. gibsoni infections occur
in dogs engaged in fighting activities.23,29 Most dogs with
B. gibsoni infection in the United States have been American pit
bull terriers.29,30 However, an increased prevalence of infection
has even been noted in fighting breeds in areas such as Japan, where
Haemaphysalis spp. are endemic. In fighting breeds, transmis-
sion has been associated with a fight or bite by an infected dog
or having been born to an infected bitch. Perinatal transmission
is believed to occur.3-5
Babesia conradae
B. conradae (previously referred to as “the California isolate”
and the “western piroplasm” and historically misidentified as
B. gibsoni)31 has been detected primarily in dogs from southern
California.31,32 Tick vectors have not been identified. Trans-
mission studies that attempted to prove vector competence for
R. sanguineus and Dermacentor variabilis were unsuccessful or
inconclusive.33 The author has identified R. sanguineus as well
as soft-bodied ticks, Ornithodoros coriaceus, on B. conradae–
infected dogs. A high prevalence of B. conradae infection was
 CHAPTER 75  Babesiosis 729

noted in a kennel of greyhound mixed-breed dogs that were

used to hunt coyotes.34 A history of coyote fights was a risk fac- Box 75-1
tor for infection. B. conradae DNA was detected in the spleen
of 1 of 50 coyotes, so fighting may be one mode of transmission
Clinical Findings in Dogs with Babesiosis
for this Babesia species.
Prevalence Duration
Babesia microti–Like Organism Common signs Hyperacute signs
A B. microti–like parasite (also referred to as Babesia annae Anorexia (primarily B. canis rossi )
or Theileria annae) has been identified in dogs, the majority of Lethargy Hypothermia
which have lived in, or traveled to, northwestern Spain.35-38 Weakness Shock
A single American pit bull terrier confiscated from a suspected Pyrexia Coma
dog-fighting operation in North America was also infected.39 Weight loss Disseminated intravascu-
A high percentage of European and North American foxes are Uncommon/atypical signs lar coagulation
infected with a genetically identical parasite.40,41 A tick vector (predominantly B. canis rossi) Metabolic acidosis
has not been definitively identified, but in Spain an association Ascites Death
between Ixodes hexagonus infestation and B. microti–like infec- Edema Acute signs
tion has been observed.42 Constipation Hemolytic anemia
Diarrhea Icterus
Feline Babesiosis Ulcerative stomatitis Splenomegaly
Feline babesiosis has not been studied as extensively as canine Hemorrhage Lymphadenopathy
babesiosis. Several small Babesia species have been identified Congested mucous Vomiting
in cats, which include Babesia felis, Babesia cati, and Babesia membranes Chronic signs
leo. Babesia felis is a highly pathogenic Babesia species that Polycythemia Intermittent pyrexia
infects domestic cats in southern Africa and the Sudan.43 Infec- Ocular and nasal discharge Partial anorexia
tion of domestic cats has primarily been identified along the Respiratory distress Loss of body condition
coast of South Africa.44,45 Affected cats are usually younger Masticatory myositis Lymphadenomegaly
than 3 years, and there is no recognized breed or sex predilec- Temporomandibular joint pain Splenomegaly
tion. Babesia cati is less pathogenic and found primarily in Back pain No signs
India. Babesia leo has been detected in lions (Panthera leo) Central nervous system signs
in Kruger National Park, as well as a single domestic cat in (seizures, ataxia, paresis)
a molecular survey.46 A small piroplasm was visualized in
blood smears from feral cats in Rio de Janeiro, Brazil, but no
molecular data were available to confirm the identity of these
organisms.47 with other tick- or bloodborne pathogens may also influence the
Large Babesia species also appear to infect cats. DNA clinical signs of disease.
sequences, most similar to those of B. canis canis and a Parasite antigens are incorporated on the erythrocyte surface
B. microti–like parasite, have been amplified from the blood of and induce host-opsonizing antibodies, which in turn leads to
three cats with retroviral infections from Spain and Portugal, removal of infected erythrocytes by the mononuclear-phagocyte
but no organisms were visualized.48 B. canis presentii was iden- system. Soluble parasite antigens can also adhere to the sur-
tified in two cats in Israel.49 Another B. canis–like parasite has face of noninfected erythrocytes and platelets. This may lead
been identified in a naturally infected cat from Poland.50 Feline to their opsonization by antibodies, with or without comple-
babesiosis has not been reported in the United States. ment, and account for hemolytic anemia and thrombocytope-
nia that is often not correlated with the level of parasitemia.
Clinical Features Parasitemia and anemia are more severe in splenectomized dogs,
and splenectomy may precipitate disease in dogs with chronic
Clinical Signs and Their Pathogenesis subclinical infections. Mechanisms other than immune-medi-
Canine Babesiosis ated destruction that contribute to erythrocyte damage include
The pathogenicity of Babesia organisms is determined primarily increased erythrocyte osmotic fragility, direct injury of erythro-
by the species and strain involved. Host factors, such as the age cytes by Babesia parasites, accumulation of cyclic nucleotides,
of the host and the immunologic response generated against the and oxidative injury.52-56 Lipid peroxidation increases eryth-
parasite or vector tick, are also important. Although most often rocyte rigidity and slows the passage of erythrocytes through
subclinically infected, dogs infected with “avirulent” species capillary beds. Soluble parasite proteases activate the kallikrein
such as B. canis vogeli may have severe clinical disease. Simi- system and induce fibrinogen-like protein formation. These pro-
larly, dogs infected with “virulent” species such as B. canis rossi teins make erythrocytes more “sticky,” and they sludge in capil-
may be subclinically infected without overt clinical or labora- lary beds, which contributes to anemia and many of the other
tory findings. However, features that should raise clinical suspi- potential clinical signs. The most severe sludging occurs in the
cion for babesiosis include fever, thrombocytopenia, hemolytic central nervous system (CNS) and muscles.
anemia, and splenomegaly. Dogs often have nonspecific signs Thrombocytopenia may result from immune-mediated
such as lethargy, anorexia, and weakness (Box 75-1). Occa- or coagulatory consumption of platelets. Despite severely
sionally owners note jaundice, mucosal pallor, or discoloration decreased platelet counts, bleeding is rarely observed in dogs
of the urine caused by bilirubinuria or hemoglobinuria. Dogs infected with most Babesia strains, and other abnormal coag-
infected with B. canis vogeli may have fever of unknown origin ulation test results are uncommon (with the exceptions of
only without overt hematologic abnormalities.51 Co-infections B. conradae and also B. canis rossi). Dogs with B. conradae
730 SECTION 4  Protozoal Diseases
infection have experienced life-threatening hemorrhages, the
pathogenesis of which requires further study.57
Other possible complications include membranoprolifera-
tive glomerulonephritis, which may have an immune-mediated
pathogenesis.58-60 Renal failure occurs in up to 40% of dogs
infected with B. microti–like parasites, and their presence has
been associated with increased mortality.35,36,38,61,62 Nonregen-
erative anemia, azotemia, and proteinuria with high urine pro-
tein/creatinine ratios were found in these dogs.35
Severe Babesiosis
Virulent B. canis canis and especially B. canis rossi strains
induce a profound systemic inflammatory response, which can
result in a severe sepsis-like syndrome with multiple organ dys-
function (see Chapter 86). The majority of dogs infected with
B. canis rossi have uncomplicated babesiosis and can be treated
as outpatients. However, up to 31% of the dogs examined at
a university clinic required hospitalization, and 10% of hos-
pitalized dogs did not survive.17 Severe clinical illness in dogs
infected with B. canis rossi results from hypotension, acute
renal failure (ARF), neurologic complications, disseminated
intravascular coagulation (DIC), a hepatopathy, and acute
respiratory distress syndrome (ARDS). Tissue hypoxia results
from anemia, shock, vascular stasis, excessive endogenous pro-
duction of carbon monoxide, parasitic damage to hemoglobin,
and decreased ability of hemoglobin to off-load oxygen.53,63
Lactic acid generation from tissue hypoxia can result in severe
metabolic acidosis.64
“Red biliary syndrome” is a paradoxical phenomenon of
severe intravascular hemolysis (manifested as hemoglobinemia
and hemoglobinuria) in combination with hemoconcentration
(high-reference-range or elevated hematocrit).63 The hemocon-
centration is thought to occur when plasma shifts from the vas-
cular to the extravascular compartment as a result of increased
capillary permeability, with a resultant decrease in blood vol-
ume. Hemoconcentration has been associated with cerebral
babesiosis, DIC, ARF, and ARDS.
Neurologic complications result from sludging of parasitized
erythrocytes in CNS capillary beds, with congestion and macro-
scopic and microscopic hemorrhages. Severe hypoglycemia can
also result in neurologic signs. Other complications of severe
babesiosis include pancreatitis, rhabdomyolysis, ocular involve-
ment, upper respiratory signs, cardiac arrhythmias, necrosis of
the extremities, and fluid accumulation. Pulmonary, CNS, and
renal complications are associated with a higher rate of mortal-
ity. Persistent lactate concentrations above 40 mg/dL are a poor
prognostic indicator for survival.65
Feline Babesiosis
Cats with babesiosis from southern Africa generally show leth-
argy, anorexia, weakness, an unkempt haircoat, and/or diar-
rhea.45 Fever and icterus are less common. Anemia can be severe
and is the underlying reason for the clinical signs. The disease is
chronic, and signs may not be apparent until a later stage of ill-
ness. Cats usually adapt to the anemia and may have only mild
clinical signs until they are stressed by handling. Complications
of the hemolytic anemia have included hepatopathy, pulmonary
edema, renal failure, CNS signs, and concurrent infections.
Physical Examination Findings
Physical examination abnormalities in most dogs with babesio-
sis consist of fever, mucosal pallor, lethargy, splenomegaly, and
FIGURE 75-2  Greyhound infected with Babesia conradae. Mucosal hemorrhages are
apparent. These resolved after treatment with atovaquone and azithromycin. (Courtesy 
Dr. Jane Sykes, University of California, Davis.)
bounding pulses. Fever is not consistently identified, because
it often waxes and wanes. Tachycardia and tachypnea may be
present in severely anemic dogs. Mucosal hemorrhages and/
or epistaxis may be present in dogs with B. conradae infection
(Figure 75-2), and excessive bleeding from venipuncture sites
may be noted.
Dogs with severe babesiosis may show CNS signs such as
incoordination, pelvic limb paresis, muscle tremors, nystag-
mus, anisocoria, intermittent loss of consciousness, seizures,
stupor, coma, aggression, or vocalization.63 Dogs with red
biliary syndrome may have congested mucous membranes or
icterus. Other clinical abnormalities in dogs with severe babe-
siosis include tachypnea, increased lung sounds, and cardiac
arrhythmias.
Affected cats may be lethargic, pale, tachycardic, and tachy-
pneic, and in some cases, icteric.
Diagnosis
Laboratory Abnormalities
Complete Blood Count
In dogs with babesiosis, the primary hematologic abnormali-
ties are anemia and thrombocytopenia. Thrombocytopenia is
often present, even when anemia is absent. A mild, normo-
cytic, normochromic anemia is generally noted in the first few
days after infection, which becomes macrocytic, hypochromic,
and regenerative as the disease progresses. Uncommonly with
B. canis rossi infections, a relative polycythemia may be
noted.63 Leukocyte abnormalities are inconsistently observed
but may include leukocytosis (with or without a left shift),
leukopenia, neutrophilia, neutropenia, lymphocytosis, and/
or eosinophilia.34,66-68 Leukopenia or a low-normal leukocyte
count due to relative neutropenia has been frequently observed
in dogs from Europe with B. canis infections and dogs from the
United States with B. conradae infections.34,68 Autoagglutina-
tion, positive direct antiglobulin (Coombs’) tests, and spherocy-
tosis may also be present.
 CHAPTER 75  Babesiosis 731

TABLE 75-1
Diagnostic Assays Available for Babesiosis in Dogs and Cats

Assay Specimen Type Target Performance

Cell culture Whole blood Babesia spp. Not widely offered or utilized for routine diagnostic
purposes. Requires several weeks’ incubation.
Cytology Whole blood, Babesia spp. Rapid and specific (i.e., when merozoites are identi-
buffy-coat smears fied by experienced cytologists, the sample is likely
(the area just below to be infected with Babesia spp.; however, Babesia
the buffy coat), spp. cannot be accurately differentiated based on
tissue aspirates morphology alone). Less sensitive than PCR.
Immunofluorescent Serum Antibodies to Acute and convalescent serology may be required
antibody serology Babesia spp. for diagnosis of acute infection, because initial
results may be negative in dogs with acute disease
and positive results may reflect previous exposure
rather than active infection. Cross-reactivity can
occur between Babesia spp. Some dogs do not
develop detectable antibody titers despite chronic
infection.
PCR Whole blood, splenic Babesia spp. DNA Confirms active infection. Sensitivity and specificity
aspirates varies depending on assay design and specimen
type. Both false-positive and false-negative results
are possible; PCR results must be interpreted in
light of the clinical signs. Serial sampling (i.e., two
or more tests on specimens obtained 2-4 weeks
apart) will increase sensitivity, especially in chroni-
cally infected animals.

In cats, anemia associated with B. felis infection is typically
macrocytic, hypochromic, and regenerative.69 Thrombocytope-
nia is an inconsistent finding.
Serum Chemistry Profile
There are no pathognomonic biochemical findings in dogs with
babesiosis. Common findings in North American dogs include
hyperglobulinemia (which may be present in the absence of
other laboratory abnormalities), mildly increased liver enzyme
activities, and, less commonly, hyperbilirubinemia. A study of
dual infections with B. canis and Ehrlichia canis showed that
the prevalence of hyperglobulinemia was higher in dogs with
dual infections than in dogs with a single infection caused by
either organism.70 Hyperbilirubinemia is a consistent finding
during acute disease caused by B. canis canis and B. canis rossi
but not by B. gibsoni or B. conradae.34,67 Dogs with severe B.
canis rossi infections may have hemoglobinemia, moderately
increased liver enzyme activities, increased BUN and serum cre-
atinine concentrations, hypoalbuminemia, hypoglycemia, and
metabolic acidosis.
Cats infected with B. felis typically have elevated serum ALT
activity and total bilirubin concentrations. Serum protein values
are usually within reference limits, but hyperglobulinemia can
occur. Renal parameters are unaffected.69
Urinalysis
Urinalysis abnormalities in dogs with babesiosis are variable but
include bilirubinuria, hemoglobinuria, proteinuria, and, in rare
cases, granular casts. Some dogs have pigmenturia consisting
most commonly of bilirubin, and occasionally hemoglobin.
Coagulation Testing
The most consistent hemostatic abnormality in complicated and
uncomplicated babesiosis is thrombocytopenia. DIC has been
reported; however, complete confirmation of DIC in animals
with babesiosis may be difficult because of the nature of the
underlying disease process and the reported unreliability of the
human fibrin degradation product test for evaluating canine
specimens. Dogs with B. conradae infection appear to have
altered platelet function tests.57
Microbiologic Testing
There are three basic methods available for specific diagnosis of
Babesia infections: microscopic identification, serologic testing,
and nucleic acid–based detection methods (Table 75-1). The true
clinical sensitivity and specificity of most of the available tests is
unknown, but all modalities can have either false-­positive or false-
negative results. Therefore, there is no “perfect” test for Babesia
infection, and in some cases, multiple diagnostic tests are indicated.
Cytologic Diagnosis
Light microscopic examination is highly specific for the identifi-
cation of Babesia organisms, but because of its limit of detection
(0.001% parasitemia), it has relatively poor sensitivity and is
not a suitable screening test. Several species/genotypes are vir-
tually indistinguishable by light microscopy, making accurate
732 SECTION 4  Protozoal Diseases
identification to the species level impossible. However, when
cytologic detection is used in combination with the history, sig-
nalment, physical examination, clinicopathologic data, and geo-
graphic location, the clinician may be able to predict the most
likely species present.
Babesia canis are large, pyriform organisms and usually exist
singly or in pairs (Figure 75-3, A). Smaller single intracellular
organisms are likely to be B. gibsoni or B. conradae (see Figure
75-3, B and C). However, when the parasites are rapidly rep-
licating, the classic intraerythrocytic forms may not predomi-
nate and some more atypical irregular, amoeboid parasite forms
with a wide range of sizes can be detected. During these phases
of infection, some small Babesia spp. have large forms and some
large Babesia spp. have small forms. In chronic infection, the
level of parasitemia is often low, especially in B. canis–infected
dogs, and thorough examination of thin blood smears is nec-
essary. Evaluation of stained slides requires experience and a
significant time commitment on the part of the laboratory tech-
nician. For B. canis infections, blood collected from the periph-
eral capillary beds of the ear tip or nailbed may yield higher
numbers of parasitized cells.71 Rarely, phagocytized organisms
and erythrocyte fragments are seen in neutrophils. Methods that
concentrate and stain buffy-coat specimens may improve the
sensitivity of parasite detection.72,73
A B
C
FIGURE 75-3  Blood smears from dog with babesiosis. A, Large unnamed Babesia piroplasm originally identified in dogs in North Carolina. A pair of large, piriform-shape merozoites
are present within an erythrocyte. B, Individual merozoites of Babesia gibsoni in erythrocytes (arrows). C, Babesia conradae merozoite within an erythrocyte (arrow). Polychromasia and
anisocytosis are also present. Although considered a small Babesia species, in this image, the organism is relatively large. Wright’s stain, 1000×.
Serologic Diagnosis
Because Babesia parasites are difficult to detect, especially in
chronic carriers, immunodiagnostics may be used to screen
for exposure. Immunofluorescent antibody (IFA) assays are
used most commonly to detect antibodies to Babesia species.
Laboratory methods differ, and each laboratory should be con-
sulted for cutoff antibody titers. As a general guideline, titers to
B. canis or B. gibsoni of 1:64 or greater on a single specimen are
supportive of exposure. Serologic assays for B. conradae are not
widely available at the time of writing.
Titers to multiple Babesia species must be measured if anti-
body testing is performed in geographic areas where more than
one species of Babesia exists. Cross-reactivity between ­Babesia
spp. makes parasite identification by PCR necessary to defini-
tively identify a given species. There are also many documented
instances in which parasites or parasite DNA have been detected
but anti-Babesia antibodies could not be detected. Very young
dogs or dogs tested early in the disease course may have nega-
tive antibody test results, so convalescent serology is required
in some cases. Antibodies were not detected in 36% of dogs
with B. canis parasitemia in one study.8 ELISAs that target sev-
eral different antigens have been developed for use in antibody
or antigen detection tests and offer promise for serodiagno-
sis of canine babesiosis in the future. However, there are no
 CHAPTER 75  Babesiosis 733

commercially available ELISA assays for the detection of anti-
Babesia antibodies in the United States.
Molecular Diagnosis Using the Polymerase Chain Reaction
Genetic methods such as PCR assays are the most sensitive and
specific means of detecting active infection with Babesia. Some
assays have a lower limit of detection that is more than 1300-fold
more sensitive than the level (0.001% parasitized erythrocytes)
of light microscopy.74 Many different PCR assays have been
published for the identification and differentiation of Babesia
infections, and several commercial diagnostic laboratories offer
real-time PCR assays for detection of Babesia infection. Species
identification can then be accomplished by DNA sequencing
or by the use of species-specific PCR assays. PCR assays that
are designed to detect multiple Babesia spp. (i.e., a broad-range
Babesia PCR) can facilitate screening. A well-designed species-
specific assay will only detect one species, and a negative test
may lead clinicians to falsely conclude that the patient is not
infected with Babesia because the wrong species was targeted
for testing. In addition, broad-range PCR assays have been help-
ful for identification of novel Babesia species.
Pathologic Findings
The most striking pathologic findings occur in dogs that die
from severe babesiosis (usually associated with B. canis rossi).
These include staining of tissues with hemoglobin or bilirubin,
hepatosplenomegaly, lymphadenomegaly, and kidneys that are a
dark-reddish color. Edema and hemorrhage, which may indicate
vascular injury and poor tissue oxygenation in severely affected
dogs, are often most severe in the lungs. Nonparasitized cells
often line the endothelial surface with parasitized cells sludged
in the lumen. Pathologic changes in the brain of these dogs
include congestion, macroscopic and microscopic hemorrhages,
sequestration of parasitized erythrocytes in capillary beds, and
pavementing of parasitized cells against the endothelium. Car-
diac histologic changes include hemorrhage, necrosis, inflam-
matory infiltrate, and fibrosis. Microthrombi of many tissues
may be evident in animals exhibiting signs of DIC. Impression
smears of the spleen may substantiate the diagnosis of babesiosis
at necropsy. Nonspecific findings in dogs with all forms of babe-
siosis include erythroid hyperplasia in the bone marrow, extra-
medullary hematopoiesis of the liver and spleen, mononuclear
phagocyte system hyperplasia, and centrilobular necrosis of the
liver. Vasculitis has been observed in B. ­conradae infections and
is associated with hepatitis and lymphadenitis with multifocal
deposits of IgM in inflamed arteries and renal glomeruli.60 In
chronic canine babesiosis and feline babesiosis, the only gross
finding may be splenomegaly.
Treatment and Prognosis
Antiprotozoal Treatment
Dogs
Dogs generally show clinical improvement within 24 to 72
hours of treatment with antibabesial drugs, but some animals
take as long as 7 days to respond (Table 75-2). Imidocarb

TABLE 75-2
Selected Antibabesial Compounds Used in the Treatment of Dogs and Cats

Organism
Interval Duration Babesia Babesia Babesia Babesia
Generic Name* Dose (mg/kg) Route (hours) (days) canis gibsoni conradae felis
Imidocarb 5-6.6 IM Once Repeat in 14 +++ + + —
dipropionate† 7.5 IM Once NA
Diminazene 3.5-5 IM Once‡ NA +++ ++ ? +
aceturate
Azithromycin§ 10 PO 24 10 +++ +++ +++ ?
and
Atovaquone§ 13.3 PO 8 10
Clindamycin‖ 25 PO 12 90 + + ? ?
and
Doxycycline 5 PO 12 90
and
Metronidazole 15 PO 12 90
Primaquine 0.5 PO 24 1-3 ? — ? +++
phosphate 1 mg per cat# IM 36 6 — — ? +++

+++, Very good; ++, good; +, fair to poor; —, not effective; ?, unknown.
*For specific information on each drug, see Chapter 10.
†For combination therapy, a dose of 3.5 mg/kg diminazene has been followed by a dose of 6.0 mg/kg imidocarb.
‡Not available in the United States, except by compassionate use. For B. canis, this dose is sufficient; for B. gibsoni, repeat dose in 24 hours. Total

dosages of 7 mg/kg or higher are associated with an increased risk of neural and parasympathomimetic toxicity.
§These two drugs should be used in combination. Atovaquone must be given with a fatty meal to facilitate absorption. The suspension causes minimal

gastrointestinal side effects.

‖Anecdotal evidence for activity against B. canis. Not effective in clearing the infection. Not recommended as sole therapy because of the potential to

develop resistance. Further documentation needed to establish efficacy of this combination (see text).
#Note that 1 mg/kg is a lethal dose.
734 SECTION 4  Protozoal Diseases
dipropionate is active against B. canis.75 Imidocarb can elimi-
nate B. canis, eliminates infection in ticks that engorge on
treated animals for up to 4 weeks after treatment, and prevents
infection for up to 6 weeks after a single injection.76 A single
dose of 7.5 mg/kg or a single dose of 6 mg/kg given the day after
a dose of diminazene (3.5 mg/kg) also clears infection.77 In areas
where reinfection is likely, some practitioners use a lower dose,
2 mg/kg SC once, that does completely clear infection. This
approach is an attempt to induce a state of premunition. Pre-
munition is the immunity of existing infection in which chronic
subclinically affected carriers may be resistant to reinfection or
at least have reduced morbidity with new infections. In contrast,
animals that have been cleared of infection are more susceptible
to reinfection with recurrent clinical disease. The risk of this
approach is that clinical babesiosis may relapse in some chronic
carriers. Therefore, this approach is not recommended for the
treatment of canine babesiosis in many parts of the United
States, where vector transmission and risk for reinfection are
thought to be low. Imidocarb does not clear B. gibsoni infec-
tions, but reduces morbidity and mortality. Therefore, this is
a reasonable alternative treatment for B. gibsoni if the owner
cannot immediately afford more effective treatments. The same
may be true for B. conradae infections. Pretreatment with atro-
pine (0.5 mg/kg SC 30 minutes before injection) reduces the
adverse effects of imidocarb (see Chapter 10).
Other related aromatic diamidines include diminazene ace-
turate, phenamidine isethionate, and pentamidine isethionate
(see Chapter 10). Although these are not available for treatment
of dogs in the United States, the first two of these drugs have
been used in many other countries and are highly active against
B. canis. They do not clear B. gibsoni infections, but can reduce
morbidity and mortality.
Atovaquone and azithromycin combination therapy is
the most effective treatment for B. gibsoni and B. conradae
infections (see Table 75-2).78,79 Atovaquone and azithromy-
cin are given for 10 days. Atovaquone must be administered
with a fatty meal to maximize drug absorption. The treat-
ment appeared to sterilize B. gibsoni infections or reduce the
parasitemia below detectable limits in approximately 80% of
dogs in a randomized double-blind placebo-controlled trial.79
However, in another study, this combination only eliminated
B. gibsoni infection (or reduced parasitemia below detect-
able limits of PCR assay), in 2 of 7 dogs.80 It also did not
clear experimental infection with two Australian isolates of
B. gibsoni.81 When atovaquone is used alone to treat B. gib-
soni infection, recrudescence of infection develops more than
30 days after treatment.81 Drug resistance has been identified
in vitro, so atovaquone should always be used in combination
with other drugs. Mutations in the genes that encode the puta-
tive molecular target of atovaquone have been identified in
B. gibsoni isolated from dogs that failed to clear infection after
atovaquone treatment.80,82
In this author’s experience, dogs that fail to clear B. gibsoni
infections after initial treatment with atovaquone and azithro-
mycin will not clear their infection after a follow-up treatment
with the same drugs, although clinical disease may resolve.
There are also two commercially available formulations of
atovaquone: a single-drug liquid suspension that is very well
tolerated, and a combination tablet product (atovaquone and
proguanil hydrochloride). Although the combination product
is less expensive, it causes severe vomiting in some patients.
Because drug absorption is critical and resistance to atovaquone
can develop or be selected, this author recommends only use
of the suspension. Dogs that have been splenectomized before
treatment do not appear to clear infections with treatment.83
Likewise, immunosuppressive therapy may reduce the ability of
antibabesial treatment to clear dogs of B. gibsoni infection. The
recommended follow-up after treatment with atovaquone and
azithromycin is a minimum of two PCR tests approximately 60
and 90 days after completing treatment.
An alternative treatment strategy can be used to treat
B. gibsoni infections that fail to respond to atovaquone and
azithromycin.84 This regimen involves a combination of
clindamycin, metronidazole, and doxycycline for a minimum
of 3 months (see Table 75-2). In an uncontrolled study, 3 of
4 experimentally infected dogs had negative PCR assay results
after treatment, whereas 1 dog remained persistently infected
and experienced clinical relapses during treatment.84 Clearance
of the infection as determined by PCR was not detected until
30 and 72 days after completing a 90-day treatment course in
2 dogs. The PCR test result became negative in the third dog
12 days into treatment. Three of the dogs were initially treated
with three doses of diminazene aceturate. In another study, 3
naturally infected dogs that had clinical relapse after or during
atovaquone and azithromycin treatment had clinical recoveries
after treatment with the clindamycin, metronidazole, and dox-
ycycline combination.80 The small number of cases reported
and the high degree of variability in the PCR results after treat-
ment make the efficacy of this combination uncertain. How-
ever, as with the atovaquone-azithromycin combination, PCR
testing should be performed 60 and 90 days after completing
therapy.
Aggressive supportive care and monotherapy with clinda-
mycin (25 mg/kg PO q12h for 7 to 21 days) has been recom-
mended if specific antibabesial drugs are not available. At this
dosage, clinical abnormalities resolve even if organism clearance
does not occur. This author does not recommend this practice
for B. gibsoni–infected dogs, because it has the potential to
select for macrolide resistance and interfere with subsequent
atovaquone and azithromycin treatment.
Other treatments that have been used to treat B. canis infec-
tions are quinuronium sulfate and 1% trypan blue solution.
Trypan blue does not clear infections and results in bluish dis-
coloration of tissues and plasma.
Cats
Treatment of feline babesiosis has not been as critically evaluated,
but most antibabesial drugs appear ineffective. Primaquine phos-
phate, an antimalarial compound, administered orally or as an
intramuscular injection, is currently the drug of choice (see Table
75-2). However, the effective dose, 0.5 mg/kg, is very close to
the lethal dose (1 mg/kg). In experimental studies, rifampin and
trimethoprim-sulfadiazine were not as effective as primaquine.85
Supportive Care
Other supportive care measures that may be required to treat
animals with babesiosis are packed red blood cell transfusions
and intravenous crystalloid fluid therapy. The administration
of the plasma component of whole blood is unnecessary in the
majority of dogs with babesiosis and can place the patient at
risk of volume overload. If rehydration is required, crystalloid
replacement solutions are preferable. Whether glucocorti-
coids are indicated to counteract the immune-mediated com-
plications of babesiosis is controversial. In one study, 21% of

134 dogs with B. canis rossi infection had hemolytic anemias
that did not respond to antibabesial therapy alone.63 This
seems less common with B. canis vogeli or B. gibsoni infec-
tions, because these often respond completely to antibabesial
treatment alone. In most dogs that require glucocorticoids,
the glucocorticoid dosage can be tapered and discontinued
within a 2- to 3-week period. Continued glucocorticoid ther-
apy may predispose the animals to other infections and has
the potential to induce ­babesial relapse.86
Immunity and Vaccination
The duration of protective immunity against B. canis infec-
tion is limited. Antibody titers may gradually decline between
3 and 5 months after infection.76 Recovered dogs are protected
against homologous infection within 5 to 8 months after infec-
tion.76 Cross-protection between strains does not occur, and
seropositivity does not guarantee protection against heterolo-
gous challenge.
A vaccine produced from cell-culture–derived exoantigens
of B. canis canis is available in Europe.87 An efficacy of 70%
to 100% has been reported, with the disease occasionally seen
in the vaccinates generally being mild. Other field studies have
been less impressive. Vaccination does not prevent infection
but appears to block initiation of many of the pathologic
processes involved in disease pathogenesis.88,89 Vaccines may
limit parasitemia and reduce development of anemia and sple-
nomegaly. A bivalent vaccine derived from soluble parasite
antigens from B. canis canis and B. canis rossi (Nobivac Piro,
MSD Animal Health South Africa) reduces clinical signs after
challenge with both species.90,91 Differences in strain antige-
nicity limit the usefulness of the commercial vaccine in other
areas. Because B. canis vaccines do not cross protect against all
B. canis subspecies, they are highly unlikely to protect against
other Babesia species.
Prevention
Treatment of babesiosis is expensive and may be ineffective, so
prevention is of paramount importance. Preventive measures
alone may be sufficient to control B. canis outbreaks in kennels
in the southeastern United States. The primary means of preven-
tion is tick control (see Prevention section in Chapter 28). Early
tick identification and removal is important because it likely
takes a minimum of 2 to 3 days of feeding for transmission of
the parasite to occur. Dogs should be tested, treated if neces-
sary, and quarantined before being introduced into a colony.
The use of amitraz-impregnated collars significantly reduced
new infections in a B. canis rossi–endemic area. In this study
none of 20 dogs treated with the collars acquired infections,
compared to 27% of 30 control dogs that received no tick pre-
vention.92 Prevention of aggressive interactions with other dogs
(especially fighting breeds such as American pit bull terriers)
may also prevent infection. All prospective canine blood donors
should be tested for babesiosis with serology and PCR assays,
CHAPTER 75  Babesiosis 735
and animals positive by either or both methods should not be
used for blood donation.
Of 16,000 greyhounds adopted through rescue leagues in
1995, 20% to 60% were seropositive for B. canis. Much of
this screening was done before the availability of PCR assays,
and serologic testing results likely overestimate the true preva-
lence of infection. The likelihood that an adopted greyhound
will develop clinical babesiosis or directly transmit infection to
other dogs is low. However, the risk to other dogs is great if the
infected animal is placed in a breeding kennel in which dogs are
housed together and tick control is not adequate, or if the ani-
mal is used as a blood donor. Treatment with imidocarb dipro-
pionate may eliminate the B. canis carrier status. This approach
should be considered in situations in which risk of spread is
likely. In other situations, the owner should be made aware of
the seropositive status so that should clinical signs consistent
with babesiosis arise, the attending veterinarian can be alerted
to the possibility of the disease. Splenectomy should be avoided
if possible in these dogs should it become indicated.
Public Health Aspects
Canine and feline Babesia spp. do not appear to pose a zoonotic
risk to immunocompetent humans. Their risks to immunocom-
promised individuals are unknown, but as with many infectious
diseases, people who are undergoing chemotherapy, are infected
with HIV, or have been splenectomized should exercise special
caution when handling blood samples from dogs. Babesiosis
is a rare tick-borne zoonosis of people in Europe and in the
United States, and isolated uncharacterized Babesia infections
have been reported in Africa and Mexico.93 The majority of
infections are mild or asymptomatic; however, some result in
severe illness and death. Splenectomized people or those older
than 55 years are especially at risk.94,95 No Babesia species has
been identified that is host specific for people. Sylvan cycles with
wild animal reservoirs occur in nature. People serve as acciden-
tal hosts for Babesia of animals when they are bitten by infected
ticks. Transmission through blood transfusion has also been
described. B. microti is the primary parasite that infects people
in the northeast and upper Midwest of the United States. The
vector tick is Ixodes scapularis, which can co-transmit anaplas-
mosis and Lyme borreliosis. The hemolytic disease with flu-like
symptoms is usually mild and self-limiting or easily treated with
clindamycin and quinine. Both asymptomatic infections and
clinical babesiosis with severe anemia have been reported in
people in California and Washington and is caused by the more
recently described parasite Babesia duncani.96-98
Babesia divergens causes a severe form of human babesio-
sis in Europe, which usually occurs in people who have had a
splenectomy and is often fatal. Babesiosis was also identified in
splenectomized people from Italy and Austria with a new strain
(EU1) that was more closely related to Babesia odocoilei.98
Identical isolates of Babesia have been found in Ixodes ricinus
ticks from Slovenia, suggesting a more widespread distribution
of this organism in Europe.
736 SECTION 4  Protozoal Diseases

CASE EXAMPLE Pretreat-

ment
Posttreat- Reference
ment Range
Bartonella <1:16 ND
Signalment: “Dora,” a 4-year-old female spayed American
­vinsonii IFA
Staffordshire terrier from North Carolina
Bartonella <1:16 ND
History: Dora was evaluated for a 3- to 4-day history of anorexia
­henselae IFA
and lethargy. Before referral, an ELISA that detects heartworm
Rickettsia 1:64 ND
antigen and antibodies to E. canis or B. burgdorferi antigens
­rickettsii IFA
was performed and the results were negative. The dog had
SNAP 4Dx ELISA Negative for ND
mild anemia (HCT 28.4%) and moderate thrombocytopenia
all agents
(59,000 platelets/µL) on an automated hematology analyzer.
Babesia spp. PCR Positive Negative
Amoxicillin–clavulanic acid, doxycycline, and famotidine
(B. gibsoni)
were prescribed. The patient began showing signs of
increased energy and appetite as well as an increase in the
HCT (38%) and platelet count (147,000 platelets/µL) after 4 Diagnosis: Babesia gibsoni infection.
weeks of treatment. However, because the values did not Treatment: Atovaquone and azithromycin.
return to within reference range, the case was referred. Comments: Dogs with babesiosis often have a positive
Physical Examination: clinical response to doxycycline, which leads clinicians to
Body Weight: 24.2 kg. suspect rickettsial infection. Although fever is considered
General: Bright, alert, responsive, hydrated. T = 101.6°C a classic sign of babesiosis, most dogs are afebrile during
(38.7°F), HR = 100 beats/min, RR = 24 breaths/min, mucous initial examination, and waxing and waning fever is
membranes pink, CRT <2 s. Physical examination was often documented with serial rectal temperatures.
otherwise unremarkable. Thrombocytopenia is often a more prominent hematologic
Selected Laboratory Findings: finding than anemia and may be the only finding in some
cases. Babesiosis may be associated with changes in both
Pretreat- Posttreat- Reference albumin and globulin concentrations. Hypoalbuminemia
ment ment Range can be secondary to proteinuria (not documented in this
PCV (%) 41 52 39-58 case). Globulins are often increased, although they may not
Platelet count 80 361 190-468 be above the high end of the reference range. Organisms are
(× 103/µL) frequently not observed on routine examination of stained
TP (g/dL) 6.3 6.5 5.2-7.3 blood smears, and PCR assay is required to document
Albumin (g/dL) 2.7 3.8 3-3.9 infection. Panels that detect antibodies to tick-borne
Globulin (g/dL) 3.5 2.7 1.7-3.8 pathogens typically either do not include Babesia spp.
Albumin/globulin 0.78 1.38 0.9-1.8 at all or only detect antibodies against B. canis. Serologic
ratio cross-reactivity between species can occur, leading to
Visualization of Negative Negative inappropriate diagnosis and treatment recommendations.
Babesia on The positive titer to Rickettsia spp. was interesting, but
microscopic in this geographic location, approximately 20% to 25%
examination of of healthy dogs are seropositive to Rickettsia spp., so its
blood smear significance was unclear. Although acute phase serology
Babesia canis IFA 1:256 ND for B. gibsoni was not done, a convalescent titer to B.
Ehrlichia canis <1:16 ND gibsoni at a follow up examination was 1:64.
IFA

SUGGESTED READINGS
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Di Cicco MF, Downey ME, Beeler E, et  al. Re-emergence of Babesia
conradae and effective treatment of infected dogs with atovaquone
and azithromycin. Vet Parasitol. 2012;187:23-27.
Jefferies R, Ryan UM, Jardine J, et al. Babesia gibsoni: detection during
experimental infections and after combined atovaquone and azithro-
mycin therapy. Exp Parasitol. 2007;117:115-123.
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CHAPTER 76
Cytauxzoonosis
Leah A. Cohn
Overview of Cytauxzoonosis
First Described: Cytauxzoon felis was first described in domes-
tic cats from Missouri in 19761
Cause: Cytauxzoon felis, a hemoprotozoan parasite (phylum
Apicomplexa)
Affected Hosts: Any felid; bobcats are the major reservoir
host.
Mode of Transmission: Amblyomma americanum (Lone Star
tick), possibly Dermacentor variabilis (American dog tick)
Geographic Distribution: South central, southeastern, and
mid-Atlantic United States, and South America
Major Clinical Signs: Lethargy, anorexia, dyspnea, fever,
icterus, anemia
Differential Diagnosis: Hemotropic mycoplasmosis, tularemia,
cholangiohepatitis, sepsis, primary immune-mediated
hemo­lytic anemia, oxidative toxicities, toxoplasmosis,
feline infectious peritonitis
Human Health Significance: Cytauxzoon felis is not known to
infect humans.
Etiology and Epidemiology
Cytauxzoonosis, a life-threatening acute febrile illness of cats,
is caused by the hemoprotozoan parasite Cytauxzoon felis, an
apicomplexan parasite within the order Piroplasmida and family
Theileriidae.2 The parasite infects both domestic and wild Feli-
dae, but not other mammals.3 Both Amblyomma americanum
(Lone Star ticks) and Dermacentor variabilis (American dog
ticks) harbor the pathogen and are competent transmission vec-
tors, although it is likely that Amblyomma ticks are the more
important natural disease vectors.4-6
As for other apicomplexan parasites, the parasite life cycle
involves sexual reproduction in the vector tick and asexual
reproduction in the mammalian host. The parasite is inoculated
into the host as a sporozoite when an infected tick vector feeds.
As for other members of the Theileriidae family, the pathogen
exists within the mammalian host in both a non-erythrocytic
(schizont) and an erythrocytic (piroplasm) form. Approxi-
mately 2 weeks after inoculation, schizogony occurs, whereby
mononuclear cells become distended with parasites.7,8 When
these cells rupture, merozoites are released and endocytosed by
RBCs, where they are known as piroplasms.8 The piroplasms
are ingested by naïve ticks during feeding (Figure 76-1). The
acute illness, known as cytauxzoonosis, occurs during the
schizogenous phase of infection, whereas piroplasms can be
found both during illness and in healthy carrier cats.
There is no breed, sex, or age predisposition for infection,
and retroviral infection has not been proven to predispose
cats to infection or disease.9 Often, infected cats are young
adults and previously quite healthy. They are typically out-
door or indoor/outdoor cats from wooded suburban or rural
areas, where they are more likely to encounter a tick that has
recently fed on an infected bobcat (Lynx rufus), the predomi-
nant reservoir host.9,10 Often, multiple cats in a neighborhood
are affected nearly simultaneously, which likely reflects the
presence of infected ticks. Infection occurs most commonly in
spring and summer when the tick vector is most active.9 Origi-
nally identified in only the south-central United States, the
geographic range of C. felis has expanded simultaneously with
expansion in the range of the vector Lone Star ticks. Infections
in domestic cats are now recognized throughout the south-­
central, southeastern, and mid-Atlantic states, and the patho-
gen has been identified in bobcats as far north as Pennsylvania
and North Dakota (Figure 76-2).10,11
Clinical Features
Signs and Their Pathogenesis
Owners of cats with cytauxzoonosis usually notice an acute
onset of profound lethargy and anorexia in a previously healthy
cat. Less consistently, owners report vocalization, elevation of
the nictitans, tachypnea and/or dyspnea, or icterus, or that the
cat feels warm to the touch.12 The vast majority of the con-
siderable pathologic damage that occurs during acute illness is
due to the schizogenous phase of parasitemia.8 The degree of
schizogony appears to be correlated with severity of disease;
bobcats (the natural host) undergo a brief and milder schizog-
enous phase as compared to domestic cats, and correspondingly
illness is less pronounced in bobcats than in domestic cats.13
Schizont-distended mononuclear cells occlude small veins and
capillaries, most notably in the liver, lung, spleen, and lymph
nodes.8,14 Obstruction fuels hypoxic tissue damage and release
of inflammatory cytokines, which can lead to systemic inflam-
matory response syndrome, sepsis, disseminated intravascular
coagulation (DIC), and multisystem organ failure. The presence
of piroplasms may trigger hemolysis in the later stages of the
acute illness; however, hemolysis abates in surviving cats, and
hemolytic anemia has not been noted in chronic carriers.15-18
Physical Examination Findings
Physical examination findings associated with acute cytauxzo-
onosis are nonspecific.8,12,19 The most consistent single finding
is fever. Although temperatures greater than 104°F (40°C) are
739
740 SECTION 4  Protozoal Diseases

FIGURE 76-1  Life cycle of Cytauxzoon felis. The parasite is maintained in bobcats (top) which are subclinically infected. The sexual cycle of the organism occurs in Amblyomma ameri-
canum hard ticks, which then transmit sporozoites to domestic cats. Sporozoites invade mononuclear cells, where they undergo schizogony. Schizont-filled monocytes obstruct venules and
capillaries and cause multiorgan failure and death. In cats that survive the schizogony phase, the organism infects erythrocytes and forms piroplasms. The importance of domestic cats as a
reservoir for infection of ticks is unknown. The schizogony stage in the bobcat is relatively mild and brief.

FIGURE 76-2  Geographic distribution of Cytauxzoon felis infections. Shaded areas

show confirmed cases in either domestic cats (blue), or bobcats only (ND and PA). The yel-
low region represents the distribution of Amblyomma americanum ticks.

common, body temperature drops in moribund animals, and

many cats are hypothermic for several hours before death.8,12
Cats are almost always lethargic. Icterus and pallor are com-
FIGURE 76-3  Cat with Cytauxzoon felis infection. There is elevation and hyperemia of
mon findings but may not be present initially. The nictitating the nictitating membranes.
membrane is commonly elevated (Figure 76-3). Both tachycar-
dia and tachypnea, with or without increased respiratory effort,
are also common. Cardiac murmurs are sometimes auscultated, Diagnosis
especially in anemic cats. Splenomegaly, hepatomegaly, and
lymphadenomegaly may be appreciable. Cats may have seizures Laboratory Abnormalities
or become obtunded shortly before death. Because the onset of Complete Blood Count
illness occurs as long as 2 weeks after the cat is bitten by a tick, Abnormalities on the CBC in cats with cytauxzoonosis commonly
ectoparasites are not consistently found on examination. include pancytopenia or bicytopenia and characteristic signet ring

FIGURE 76-4  Wright-Giemsa–stained peripheral blood smear from a cat with
cytauxzoonosis at 1000× magnification. Multiple RBC contain signet ring–shaped piro-
plasms (short arrow); a single RBC demonstrates a tetrad form (long arrow). (Courtesy Dr.
Marlyn Whitney, University of Missouri Veterinary Medical Diagnostic Laboratory.)
intraerythrocytic inclusions (see Cytologic Examination) (Figure
76-4). Various combinations of neutropenia, lymphopenia, throm-
bocytopenia, and nonregenerative anemia are observed.12,19,20
Occasionally, neutrophilia or bandemia are observed. Although
thrombocytopenia is usually identified and may be related to DIC
or vasculitis, platelet clumping is common even in healthy cats
and can result in false thrombocytopenia. Anemia may be absent
at the onset of illness but usually develops about the same time
piroplasms become identifiable. Because of the acute nature of the
disease, anemia is initially nonregenerative. In recovered carrier
cats, cell counts rebound to values within reference ranges, and
chronic anemia is not a feature of the carrier state.15,17,18,21
Serum Chemistry Profile
Although there are no specific findings on serum chemistry
analysis, a number of abnormalities are commonly identified in
cats with cytauxzoonosis. Hyperbilirubinemia is common and
may result from hepatic vascular occlusion and/or hemolysis.
The activities of serum ALP and ALT increase in a minority of
sick cats, and increases are usually mild to moderate.12 Mild to
moderate hyperglycemia is common in stressed, sick cats. Mild
hypoproteinemia, hypocalcemia, hyponatremia, and hypokale-
mia can occur during acute illness.12,19 No consistent abnor-
malities have been reported on serum chemistry analysis for
recovered carrier cats.17,21
Urinalysis
Urinalysis results are rarely reported for cats with cytauxzoono-
sis.19 Certainly, hyperbilirubinemia may be reflected by bilirubi-
nuria, and hyperglycemia that surpasses the renal threshold may
cause mild glucosuria. In the author’s experience, the urinalysis
is otherwise unremarkable.
Clotting Function
Acute cytauxzoonosis is a form of sepsis and, as such, is expected
to be associated with altered coagulability. Thrombocytopenia
CHAPTER 76  Cytauxzoonosis 741
is common in affected cats, but because platelet clumping occurs
readily in cats, the frequency of true thrombocytopenia may be
overestimated.12,19,20 Although not often assayed, prolongation
of APTT and PT in several affected cats suggests that DIC is a
common complication.22,23
Diagnostic Imaging
Imaging studies do not contribute directly to the diagnosis of
cytauxzoonosis, and results are seldom reported in the literature.
Splenomegaly, hepatomegaly, or both may be identified either
on plain radiographs or on abdominal ultrasound examination.
Although thoracic radiographic findings are seldom documented
in the literature, the author has commonly identified diffuse
interstitial pulmonary patterns on radiographs from cats with
respiratory signs. Less commonly, alveolar lung patterns or pleural
effusion are recognized.
Microbiologic Testing
Diagnostic assays currently available for cytauxzoonosis in cats
are described in Table 76-1.
Cytologic Examination of Aspirates or Blood Smears
Careful microscopic examination for intracellular parasites is
the most commonly used method for diagnosis of infection. A
well-made thin blood smear stained with Wright’s stain or Diff
Quik should be carefully scanned for either RBC piroplasms
or schizont-laden mononuclear cells. Schizont-distended mono-
nuclear cells are sometimes, but not usually, found on careful
examination of the entire feathered edge of the blood smear
during acute illness (Figure 76-5). If these cells are identified,
a diagnosis of acute cytauxzoonosis is confirmed. More com-
monly, microscopic review allows identification of piroplasms
within RBCs. Unfortunately, because illness is most associated
with schizogenous replication, illness precedes development of
piroplasms in up to half of cats examined. When cytauxzoono-
sis is suspected but piroplasms are not identified, reexamination
of another blood smear the following day may yield positive
results. Piroplasms are most often 1 to 1.5 µm, signet ring–
shaped inclusions, but tetrad, “safety pin,” and coccoid organ-
isms are seen on occasion (see Figures 76-4 and 76-5).8 Stain
precipitate, Howell-Jolly bodies, or other RBC pathogens such
as Mycoplasma haemofelis have features that distinguish them
from C. felis piroplasms, but a less experienced microscopist
may misidentify them as C. felis. Importantly, recovered cats
may harbor piroplasms for months to years after initial infection.
It is important to realize that piroplasms might be discovered
incidentally in chronic carrier cats that survived infection in the
distant past. Thus, although identification of piroplasms con-
firms infection, piroplasms alone (especially in low numbers)
cannot confirm that current illness is due to cytauxzoonosis.
When compared with peripheral blood, mononuclear cells
distended with merozoites are more commonly identified on
fine-needle aspiration (FNA) from spleen, liver, lymph nodes, or
lungs of affected cats. Because schizogony precedes piroplasm
development, FNA of these organs can rapidly confirm a diag-
nosis when piroplasms are absent (Figure 76-6).
Molecular Diagnosis using the Polymerase Chain Reaction
PCR assays are the most sensitive diagnostic test for cytauxzoono-
sis.18,24 Several commercial veterinary diagnostic laboratories that
perform molecular diagnostics offer PCR analysis of whole blood
samples for Cytauxzoon felis DNA. As with any PCR test, only
742 SECTION 4  Protozoal Diseases

TABLE 76-1
Diagnostic Assays Currently Available for Cytauxzoonosis in Cats
Assay Specimen Type Target Performance
Cytologic examination Whole blood smears, Cytauxzoon felis piroplasms in Piroplasms may initially not be visible in
aspirates of lymph nodes, erythrocytes, or merozoites/ acutely ill cats or may be confused with
spleen, liver, lung schizonts in mononuclear cells other erythrocyte inclusions such as
or reticuloendothelial tissues hemoplasmas or Howell-Jolly bodies.
Schizonts may be found in fine-needle
aspirates of reticuloendothelial tissues
before piroplasms are found on blood
smear examination in some cats.
PCR assays Whole blood C. felis DNA Sensitivity and specificity may vary
depending on assay design, but PCR
assays may be more sensitive than
cytology for detection of piroplasms,
especially in chronic carrier cats.

FIGURE 76-5  Wright-Giemsa–stained peripheral blood smear from a cat with

cytauxzoonosis at 1000× magnification. A mononuclear cell massively distended with a
schizont is surrounded by multiple RBCs, a few of which contain Cytauxzoon felis piroplasms.

reputable laboratories should be used; quality control is crucial.
Because of the delay inherent in any mail-out PCR test, presump-
tive disease treatment needs to precede the results of PCR testing.
Healthy, chronic carrier cats have been identified by PCR
assays applied to peripheral blood samples.18,25,26 Although the
importance of such carrier cats in propagation of infection is
not known, they are competent hosts for transmission of infec-
tion to other cats via the bite of a tick vector.27 Some cat owners
may request screening of healthy cats that reside in the same
neighborhood as a cat with cytauxzoonosis; such screening is
best accomplished with PCR assays. Unfortunately, no method
has been confirmed to completely eliminate parasitemia from
chronic carrier cats.21,28,29
Other Diagnostic Assays
To date, C. felis has not been successfully cultured in  vitro.
Although serologic tests have been developed for detection of
antibodies to C. felis, they are not practical for disease diagnosis,
because illness likely precedes antibody formation.30
FIGURE 76-6  Fine-needle aspirate of a lymph node from a cat with cytauxzoonosis.
Large numbers of merozoites (which have a granular appearance) erupt from a mono-
nuclear cell.
Pathologic Findings
Gross Pathologic Findings
Icterus and pallor are common gross pathologic findings in cats
that die from cytauxzoonosis.8,19,31 The spleen and liver are
often enlarged, and mesenteric lymph nodes may be enlarged
and edematous.8,19,31 The lungs are often wet and fail to col-
lapse. Pleural effusion is sometimes identified, as is pericardial
effusion.32 Petechial hemorrhages are occasionally found on the
serosal surface of organs.8,33
Although histopathology is not a commonly performed ante-
mortem diagnostic test, a diagnosis of cytauxzoonosis is easily
confirmed at necropsy by histopathology.19,31,33 Numerous
venules and capillaries, especially in the spleen, liver, and lungs,
are occluded with markedly enlarged merozoite-distended
mononuclear cells (Figure 76-7).19,33,34 The germinal centers of

FIGURE 76-7  Histopathologic image of a section of spleen from a cat that died of
cytauxzoonosis. Schizont-distended mononuclear cells obstruct blood vessels. H&E stain,
400× magnification. (Courtesy Dr. Linda Berent, University of Missouri Veterinary Medical
Diagnostic Laboratory.)
lymphoid tissues contain many parasitized mononuclear cells
as well, but organisms are not found in lymphocytes them-
selves.8 Pulmonary lesions, which are common, are typified
by moderate to severe vascular occlusion of medium-caliber
vessels and interstitial pneumonia with edema and neutrophil
accumulation.35
Treatment and Prognosis
Antiprotozoal Drugs
Currently, the treatment of choice for feline cytauxzoonosis
is a combination of atovaquone suspension (Mepron, Glaxo-
SmithKline, Research Triangle Park, NC) and azithromycin
(see also Chapter 10) (Table 76-2). Both drugs are adminis-
tered orally for 10 days.12 Although atovaquone is expensive,
a single bottle will treat many cats. Compounding pharmacies
may supply reasonably priced atovaquone in smaller quanti-
ties. In recovered carrier cats, the combination of atovaquone
and azithromycin does not consistently eliminate parasitemia,
but in most treated cats, pathogen burden drops to levels below
PCR detection and piroplasms are no longer visible on periph-
eral blood smears.19
Other antiprotozoal therapies have been investigated for the
treatment of cytauxzoonosis. Parvaquone and buparvaquone,
both used to treat related Theileria infections, were ineffective
for treatment of experimental cytauxzoonosis.36 Imidocarb
dipropionate has not proved to be an effective treatment for
cytauxzoonosis; only 25% of cats treated with the drug sur-
vived.12 Additionally, imidocarb does not reduce the parasite
burden in carrier cats.29 Diminazene aceturate is a drug used
extensively in Africa, South America, and the far East for treat-
ment of protozoal diseases. In a retrospective report, 5 of 6 cats
treated with diminazene aceturate at 2 mg/kg IM, plus support-
ive care, survived acute cytauxzoonosis.22 The author has used
diminazene to treat four additional cats with cytauxzoonosis,
and only one survived. Studies in naturally infected cats are
difficult to conduct because this drug is not approved for use
in the United States. As with imidocarb, diminazene aceturate
was unable to eliminate or reduce parasitemia from healthy
carrier cats.21,30
CHAPTER 76  Cytauxzoonosis 743
TABLE 76-2
Antimicrobial Drugs Indicated for Treatment of Feline
Cytauxzoonosis
Dose Interval Duration
Drug (mg/kg) Route (hours) (days)
Atovaquone/azithro-
mycin combination:
Atovaquone and 15 PO 8 10
Azithromycin 10 PO 24 10
Supportive Care
Cats with cytauxzoonosis are critically ill and require support-
ive care. Crystalloid fluids address dehydration, correct prerenal
azotemia, and may improve organ perfusion but should be used
judiciously in cats with evidence of pulmonary damage or pleural
effusion. Whole blood or packed RBC transfusion is often necessary
during the hemolytic stage of acute illness. Plasma administration
may be useful in cats with DIC, and although the recommendation
is controversial, some clinicians advocate treatment with heparin
at 200 units/kg SC q8h.12 Because affected cats may be severely
febrile and apparently in pain, antipyretic, anti-inflammatory,
and analgesic drugs are often used. Unfortunately, no controlled
studies have examined whether these therapies improve or worsen
clinical outcome. The author does not usually administer either
glucocorticoids or nonsteroidal anti-inflammatory drugs but has
treated cats with buprenorphine as an analgesic. There are numer-
ous reports of antibacterial drugs used in the therapy of cats with
cytauxzoonosis, but the drugs utilized (enrofloxacin, doxycycline,
and/or sodium ampicillin) seem unlikely to have contributed sub-
stantially to any improved outcome.19,37 Cats typically have a poor
appetite during recovery from cytauxzoonosis. Early placement of
a nasoesophageal or esophagostomy feeding tube not only allows
for provision of enteral nutrition and hydration, but facilitates
administration of oral medications (i.e., atovaquone and azithro-
mycin) with minimal stress to the patient.
Prognosis
In the early history of the disease, treatment options for cytaux-
zoonosis were considered ineffective and the disease was
regarded as uniformly fatal. However, it is now recognized that
some cats become infected without recognized clinical illness,
and others survive illness with supportive or specific medical
care.12,15,18,26,38 Either differences in the individual cat’s immune
response to infection or reduced pathogenicity of the infecting
organism are possible explanations for chronic infection in cats
with no history of prior illness. The latter theory is supported
by finding many cats with chronic infection but no history of
illness in a limited geographic region around northwestern
Arkansas.15,27,39
Despite occasional recognition of subclinically infected cats,
most infected cats become extremely ill and die in the absence of
aggressive treatment.19,39 Even when treated with atovaquone
and azithromycin, only 60% of infected cats survive.12 Because
most cats that die despite therapy do so within a day of pre-
sentation, even the most active antiprotozoal compound may
not prevent death in a proportion of infected cats once clinical
illness is recognized.
744 SECTION 4  Protozoal Diseases
Immunity and Vaccination
Cats that survive experimentally induced schizogenous infection
appear to be immune to illness on re-infection, although experi-
mental inoculation with piroplasms alone does not provide
protection.36,40,41 Resistance to re-infection with C. felis offers
hope that successful vaccine development may be possible, but
safe and effective vaccines against apicomplexan parasites are
notoriously difficult to develop.42 Inoculation of cats with a
less pathogenic Cytauxzoon species (C. manul) failed to reduce
mortality associated with C. felis challenge.43 Many veterinar-
ians in endemic regions have observed clinical illness indistin-
guishable from cytauxzoonosis in cats known to have survived
cytauxzoonosis in previous years. Because no attempt has been
made to look for schizonts in the cats experiencing a second ill-
ness, it is unclear if these cats have recrudescent cytauxzoonosis,
re-infection, or some other illness altogether.
Prevention
For now, prevention relies on avoidance of tick exposure. Given
the sensitivity of domestic cats to many commonly used ecto-
parasiticides, the arsenal of acaricides available for cats is lim-
ited to flumethrin-impregnated collars, fipronil, and possibly
CASE EXAMPLE
Signalment and History: “Ginger,” a 2-year-old female
spayed domestic longhaired cat that lived near Springfield,
Missouri, was evaluated for a 1-day history of anorexia and
lethargy in April of the year. The cat was an indoor/outdoor
cat with no known history of tick exposure. There were no
other cats in the household.
Physical Examination: On examination, the cat’s rectal
temperature was 105.6°F (40.9°C), with a heart rate of 200
beats/min and a respiratory rate of 28 breaths/min. The
spleen was prominent on abdominal palpation and the
nictitans was elevated (see Figure 76-3), but examination
was otherwise unremarkable.
Laboratory Findings: Blood was obtained for a CBC and
retrovirus testing. The cat tested negative for FeLV antigen
and FIV antibody. The cat had a mild, nonregenerative
anemia (PCV 27%) and leukopenia (4200 cells/µL, reference
range 5500-19,500 cells/µL). Although the platelet count
was low, platelet clumps were present. Plasma in the spun
hematocrit tube was icteric. No parasites were observed on
microscopic slide review.
Because of a high degree of suspicion for cytauxzoonosis, a
fine-needle aspirate was obtained from the spleen. Multiple
markedly enlarged mononuclear cells containing numerous
merozoites were observed.
Diagnosis: Cytauxzoonosis.
Treatment and Outcome: A nasoesophageal tube
was placed to facilitate administration of atovaquone
suspension (15 mg/kg PO q8h) and azithromycin (10
mg/kg PO q24h). A cephalic vein catheter was placed
and intravenous crystalloid fluids administered at a
selamectin, although the latter does not make a label claim of
efficacy against ticks in cats.44,45 Given their potent activity
against ticks and their safety in cats, flumethrin-impregnated
collars offer the greatest potential for prevention of cytauxzo-
onosis in cats that reside in endemic areas. However, no ecto-
parasiticide prevents tick bites entirely.12,46 Where possible,
cats in endemic areas should be kept indoors to minimize tick
exposure.
Recovered cats may remain carriers, and may serve as reser-
voirs for infection of naïve ticks.6 For this reason, recovered cats
should also be kept indoors and ectoparasite control practices
instituted in order to minimize the risk of transmission to other
cats via tick bite. Treatment of recovered cats with imidocarb
dipropionate or diminazene aceturate does not reduce piro-
plasm parasitemia, but treatment with atovaquone and azithro-
mycin may reduce the parasite burden and potentially reduce
the risk of transmission to feeding ticks.29,30
Public Health Aspects
Cytauxzoon felis is not known to infect humans. However,
infected cats have the potential to be infected with other vector-
borne pathogens, so precautions should be taken to avoid needle-
stick injuries or direct skin contact with blood from infected cats.
maintenance rate. Heparin (200 units/kg SC q8h) and
buprenorphine (0.01 mg/kg SC q8h) were administered.
The following day, the cat’s PCV was 20% and icterus was
evident on physical examination. Additionally, many
piroplasms were visible on cytologic analysis of a smear
of peripheral blood. A serum chemistry profile confirmed
hyperbilirubinemia and a mild increase in the activity of
serum ALT but was otherwise unremarkable.
By day 3, the PCV was 12%, and both respiratory and heart rate
were increased. The cat was administered a single unit of
whole blood, after which the PCV increased to 19%. Nutri-
tional support was administered via the nasoesophageal
tube, but otherwise treatment remained unchanged.
On day 5, the PCV dropped to 11%, which necessitated another
whole blood transfusion. The cat remained tachypneic after
transfusion. Thoracic radiographs revealed pleural effusion
and a marked diffuse interstitial lung pattern. The cat was
treated with supplemental oxygen, and 25 mL of a clear, yel-
low, modified transudate was removed using thoracocentesis.
By day 7, the cat no longer required supplemental oxygen to
maintain respiratory function, and the anemia was regen-
erative. The cat was discharged from the hospital on day
8 with continued administration of atovaquone and azithro-
mycin at home, but all other treatments were discontinued.
On recheck examination 1 week later, the cat was seemingly
normal to the owners, and no abnormalities were detected
on physical examination. The PCV was 29%, platelet and
WBC counts had normalized, and a single piroplasm was ob-
served on careful review of the entire feathered edge of the
blood smear. The cat’s owners were advised to keep the cat
entirely indoors and to use an ectoparasiticide regularly to
minimize any risk that the cat could serve as a reservoir for
infection of ticks. The cat remained healthy 1 year later.

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azithromycin or imidocarb dipropionate in cats with acute cytauxzo-
onosis. J Vet Intern Med. 2011;25:55-60.
Reichard MV, Baum KA, Cadenhead SC, et  al. Temporal occurrence
and environmental risk factors associated with cytauxzoonosis in
domestic cats. Vet Parasit. 2008;152:314-320.
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J Wildl Dis. 1985(21):190-192.
17. Lewis KM, Cohn LA, Birkenheuer AJ. Lack of evidence for perina-
tal transmission of Cytauxzoon felis in domestic cats. Vet Parasitol.
2012;188:172-174.
18. Brown HM, Latimer KS, Erikson LE, et al. Detection of persistent
Cytauxzoon felis infection by polymerase chain reaction in three
asymptomatic domestic cats. J Vet Diagn Invest. 2008;20:485-488.
CHAPTER 76  Cytauxzoonosis 745
19. Hoover JP, Walker DB, Hedges JD. Cytauxzoonosis in cats: eight
cases (1985-1992). J Am Vet Med Assoc. 1994;(205):455-460.
20. Franks PT, Harvey JW, Shields RP, et al. Hematological findings
in experimental feline cytauxzoonosis. J Am Anim Hosp Assoc.
1988;24:395-401.
21. Lewis KM, Cohn LA, Marr H, et  al. Diminazene diaceturate for
the treatment of chronic Cytauxzoon felis parasitemia in naturally
infected cats. J Vet Intern Med. 2012;26:1490-1493.
22. Greene CE, Latimer K, Hopper E, et al. Administration of dimina-
zene aceturate or imidocarb dipropionate for treatment of cytaux-
zoonosis in cats. J Am Vet Med Assoc. 1999;215:497-500.
23. Garner MM, Lung NP, Citino S, et  al. Fatal cytauxzoono-
sis in a captive-reared white tiger (Panthera tigris). Vet Pathol.
1996;33:82-86.
24. Birkenheuer AJ, Marr H, Alleman AR, et  al. Development and
evaluation of a PCR assay for the detection of Cytauxzoon felis
DNA in feline blood samples. Vet Parasitol. 2006;137:144-149.
25. Haber MD, Tucker MD, Marr HS, et al. The detection of Cytauxzoon
felis in apparently healthy free-roaming cats in the USA. Vet Parasitol.
2007;146:316-320.
26. Lewis KM, Cohn LA, Downey M, et al. Evaluation of Cytauxzoon
felis infection status in captive-born wild felids housed in areas endemic
for this pathogen. J Am Vet Med Assoc. 2012;241:1088-1092.
27. Reichard MV, Meinkoth JH, Edwards AC, et al. Transmission of
Cytauxzoon felis to a domestic cat by Amblyomma americanum.
Vet Parasitol. 2009;161:110-115.
28. Cohn LA, Birkenheuer AJ, Ratcliff E. Comparison of two drug pro-
tocols for clearance of Cytauxzoon felis infections [Abstract]. J Vet
Intern Med. 2008;22:704.
29. Lewis KM, Cohn LA, Marr H, et al. High dose diminazene diacetu-
rate does not eliminate parasitemia in cats with chronic Cytauxzoon
felis infection. Proceedings of the American College of Veterinary
Internal Medicine Forum. New Orleans: LA; 2012.
30. Cowell RL, Fox JC, Panciera RJ, et al. Detection of anticytauxzoon
antibodies in cats infected with a Cytauxzoon organism from bob-
cats. Vet Parasitol. 1988;28:43-52.
31. Aschenbroich SA, Rech RR, Sousa RS, et  al. Pathology in
practice. Cytauxzoon felis infection. J Am Vet Med Assoc.
2012;240:159-161.
32. Butt MT, Bowman D, Barr MC, et al. Iatrogenic transmission of
Cytauxzoon felis from a Florida panther (Felix concolor coryi) to a
domestic cat. J Wildl Dis. 1991;27:342-347.
33. Wagner JE, Ferris DH, Kier AB, et  al. Experimentally induced
cytauxzoonosis-like disease in domestic cats. Vet Parasitol.
1980;6:305-311.
34. Susta L, Torres-Velez F, Zhang J, et al. An in situ hybridization and
immunohistochemical study of cytauxzoonosis in domestic cats.
Vet Pathol. 2009;46:1197-1204.
35. Snider TA, Confer AW, Payton ME. Pulmonary histopathology of
Cytauxzoon felis infections in the cat. Vet Pathol. 2010;47:698-702.
36. Motzel SL, Wagner JE. Treatment of experimentally induced
cytauxzoonosis in cats with parvaquone and buparvaquone. Vet
Parasitol. 1990;35:131-138.
37. Walker DB, Cowell RL. Survival of a domestic cat with naturally
acquired cytauxzoonosis. J Am Vet Med Assoc. 1995;206:1363-1365.
38. Brown HM, Lockhart JM, Latimer KS, et  al. Identification and
genetic characterization of Cytauxzoon felis in asymptomatic
domestic cats and bobcats. Vet Parasitol. 2010;172:311-316.
39. Brown HM, Berghaus RD, Latimer KS, et al. Genetic variability of
Cytauxzoon felis from 88 infected domestic cats in Arkansas and
Georgia. J Vet Diagn Invest. 2009;21:59-63.
40. Glenn BL, Kocan AA, Blouin EF. Cytauxzoonosis in bobcats. J Am
Vet Med Assoc. 1983;183:1155-1158.
41. Ferris DH. A progress report on the status of a new disease of
American cats: cytauxzoonosis. Comp Immun Microbiol Infect Dis.
1979;1:269-276.
42. Sharma S, Pathak S. Malaria vaccine: a current perspective. J Vector
Borne Dis. 2008;45:1-20.
746 SECTION 4  Protozoal Diseases
43. Joyner PH, Reichard MV, Meinkoth JH, et  al. Experimental
infection of domestic cats (Felis domesticus) with Cytauxzoon
manul from Pallas’ cats (Otocolobus manul). Vet Parasitol.
2007;146:302-306.
44. Bishop BF, Bruce CI, Evans NA, et  al. Selamectin: a novel
broad-spectrum endectocide for dogs and cats. Vet Parasitol.
2000;91:163-176.
45. Stanneck D, Kruedewagen EM, Fourie JJ, et al. Efficacy of an imi-
docloprid/flumethrin collar against fleas and ticks on cats. Parasit
Vectors. 2012;5:82.
46. Dryden MW. Flea and tick control in the 21st century: challenges
and opportunities. Vet Dermatol. 2009;20:435-440.
CHAPTER 77
Canine and Feline Hepatozoonosis
Nancy Vincent-Johnson
Overview of Canine Hepatozoonosis
First Described: Hepatozoon canis, India, 1905 (James)1; Hepa-
tozoon americanum, USA, 1978 (Craig and others)2
Cause: Hepatozoon canis, Hepatozoon americanum (phylum
Apicomplexa)
Affected Hosts: Domestic dogs, possibly some wild carnivores
Geographic Distribution: H. americanum, southeastern and
south-central United States; H. canis, Asia, Africa, southern
Europe, the Middle East, the Americas.
Mode of Transmission: Primarily by ingestion of tick vectors.
For H. americanum, the vector is Amblyomma maculatum;
for H. canis, the vector is Rhipicephalus sanguineus. Other
tick species (Amblyomma, Rhipicephalus, and Haemaphy-
salis spp.) may also be involved.
Major Clinical Signs: Dogs with H. americanum infections
most often show lethargy, fever, weight loss, locomo-
tory abnormalities, hyperesthesia, mucopurulent ocular
discharge, signs related to protein-losing nephropathy
such as polyuria and polydipsia. Dogs with severe H. canis
infections may show fever and lethargy.
Differential Diagnoses: Sterile or infectious meningitis, bacte-
rial discospondylitis, Lyme borreliosis, canine monocytic
ehrlichiosis, toxoplasmosis, sarcocystosis, systemic immune-
mediated diseases such as systemic lupus erythematosus
Human Health Significance: Canine Hepatozoon species are
not known to infect humans.
Etiology and Epidemiology
Apicomplexan protozoal parasites from the genus Hepatozoon
include more than 300 species that infect a wide variety of
amphibians, reptiles, birds, and mammals.3 Despite its name,
canine hepatozoonosis is not a zoonotic disease and rarely
affects the liver. Canine hepatozoonosis is caused by one of two
species of Hepatozoon currently known to cause disease in dogs,
Hepatozoon canis and Hepatozoon americanum (compared in
Table 77-1). First described in India in 1905, H. canis has long
been known as the agent that infects dogs in many regions of
the Old World, including Asia, Africa, southern Europe, and
the Middle East, and more recently has been identified in the
New World in both North and South America (Figure 77-1).1,3-20
Hepatozoon americanum causes American canine hepatozo-
onosis, which occurs in dogs primarily in the southeastern and
south-central United States. It was first confused with H. canis
in 1978 in dogs from the Gulf Coast region of Texas and has
subsequently been detected in dogs from Louisiana, Oklahoma,
Alabama, Georgia, Florida, and Tennesee.2,21-25 In 1997, it was
identified as a distinct species, H. americanum.26,27 Occasional
cases have been identified in diverse locations across the United
States, including Washington, California, Nebraska, Vermont,
and Virginia.17 Because infection is chronic, the infection may
be diagnosed in dogs with a history of travel to endemic areas.
The DNA of a Hepatozoon felis–like organism was detected in
a dog from southern California, but the clinical significance and
epidemiology of this organism are unknown.28
Recent reports indicate that H. canis also exists in the United
States, although infection with H. americanum is diagnosed
much more frequently. H. canis has been identified in dogs
from Mississippi, Louisiana, Alabama, Georgia, Oklahoma, and
Virginia and in a dog that lived in New Jersey but was born in
a Texas shelter.16,17,29 A few dogs have mixed infections with
both H. americanum and H. canis. Domestic cats can also be
infected by Hepatozoon spp., which include H. felis and possibly
H. canis or closely related species (see later section on feline
infections and Table 77-1).30,31
Because dogs or other vertebrates serve as intermediate
hosts, a vector is needed to complete the sexual phase of the life
cycle of Hepatozoon species. In the case of H. americanum, the
definitive host is the Gulf Coast tick, Amblyomma maculatum,
whereas the definitive host of H. canis is the brown dog tick,
Rhipicephalus sanguineus.32,33 In Japan, Haemaphysalis spp.
ticks have been implicated as vectors of H. canis, and in Brazil,
Amblyomma ovale and Rhipicephalus (Boophilus) microplus
are suspected vectors.34-36
As it feeds on an infected dog, the nymphal tick ingests
circulating leukocytes that contain gamonts. Within the tick’s
gut, the gamonts are freed from the cells and undergo further
development before fertilization occurs. The resulting zygote
divides through sporogony and develops into an oocyst within
the hemocoel of the tick (Figure 77-2). Oocysts mature while the
tick molts to the adult stage. In the case of H. americanum, the
larval stage of the tick can also become infected, resulting in a
nymph that contains viable oocysts capable of transmitting the
disease.37 Each mature oocyst contains hundreds of sporocysts,
with each sporocyst containing 10 to 26 infective sporozoites.
Unlike most tick-borne diseases, hepatozoonosis is not transmit-
ted through the bite of an infected tick but rather by ingestion of
an infected tick. This typically occurs when the dog grooms itself
but can also occur if the dog feeds on tick-infested prey. Once
ingested, exposure to bile in the dog’s intestinal tract results in
release of the sporozoites, which penetrate the intestinal epithe-
lial wall and are transported to the target organs and tissues,
likely within mononuclear cells.
747
748 SECTION 4  Protozoal Diseases

TABLE 77-1
Comparison of Hepatozoon canis Infection, American Canine Hepatozoonosis, and Feline Hepatozoonosis
First described
Etiologic agent
Primary vector
Route of transmission
Additional routes of
transmission
Affected mammalian hosts
Geographic distribution
Severity of disease
Major clinical signs
Common
laboratory abnormalities
Radiographic lesions
Histopathology findings
Blood smear findings
Human health significance
H. canis Infection
India, 1905
Hepatozoon canis
Rhipicephalus sanguineus
(brown dog tick)
Ingestion of tick
(not a tick bite)
Transplacental from dam to
puppy
Dogs, foxes, jackals, hyenas,
other carnivores
Africa, Middle East, Asia,
southern Europe, South
America, United States
Subclinical to mild;
occasionally severe, esp.
if immunosuppressed
Lethargy, fever, weight loss
Anemia
Extreme leukocytosis is rare
but may occur with high
parasitemia
Nonspecific; rare periostitis
No muscle lesions
“wheel-spoked” meronts
in spleen, bone marrow,
lymph nodes
Gamonts common; parasitemia
of 1%-5% is typical; rarely
see parasitemia of 5%-100%
None known
American Canine
Hepatozoonosis
Texas Gulf Coast, 1978
Hepatozoon americanum
Amblyomma maculatum
(Gulf Coast tick)
Ingestion of tick
(not a tick bite)
Ingestion of cystozoites
in transport hosts;
transplacental is likely
Dogs, coyotes, possibly bobcats,
ocelots, and other wildlife
Primarily southern and
southeastern United States
Usually severe, even in absence
of immunosuppression
Fever, pain, lameness, ocular
discharge
Marked leukocytosis (20,000-
200,000 cells/µL), anemia,
elevated ALP activity, low
glucose concentration
Common; periosteal bone
proliferation
Myositis, onion-skin cysts,
pyogranulomas, and
meronts in skeletal muscle
Rarely observed gamonts;
parasitemia is usually
<0.1%
None known
Feline Hepatozoonosis
India, 1908
Multiple poorly defined
Hepatozoon sp.
Unknown
Probably ingestion of tick
(not a tick bite)
Unknown
Domestic cats, wild felids
India, Africa, Israel, Brazil,
Spain, France, Thailand,
United States (Hawaii)
Typically subclinical unless
immunosuppressed
Nonspecific
Elevated CK activity
Not reported
Meronts in myocardium
and skeletal muscle
Parasitemia is rare; when
present usually <1% of
neutrophils
None known

In H. americanum infections, infected cells preferentially
travel to skeletal muscle, where each organism develops within
its host cell, which becomes lodged between myocytes (Figure
77-3). Concentric layers of mucopolysaccharide are laid down
by the host cell to form an “onion-skin cyst,” which may protect
the organism from the immune response (Figure 77-4). Merogony
occurs within the cyst, and on maturation of the meront, the
cyst ruptures, and merozoites are released. This elicits a severe
inflammatory response, with recruitment of neutrophils and
monocytes to the area. A pyogranuloma develops in the space
where the cyst existed (Figure 77-5). Many of these inflammatory
cells become infected with a single zoite. Angiogenesis within the
pyogranuloma results in a highly vascular structure from which
the infected cells can reenter circulation. The intracellular para-
sites can travel to other target sites, where they continue the
asexual reproductive cycle, or they may become gamonts. The
gamonts are then ingested by feeding ticks, which completes the
life cycle. Some of the onion-skin cysts lie dormant for varying
lengths of time; their activation is responsible for the waxing
and waning nature of the disease as well as the relapse of clinical
signs that can occur months after perceived clinical cure.
In H. canis infections, infected cells are carried by lymph
or blood to the spleen, bone marrow, lymph nodes, and other
organs such as the liver, kidney, and lungs where the organisms
divide asexually through merogony (Figure 77-6). Two types
of meronts form: the “wheel spoke” meront, which contains
20 to 30 micromerozoites which form around a central round
structure (Figure 77-7), and a meront that contains up to four
macromerozoites.38 Once released from the mature meront,
micromerozoites invade neutrophils and monocytes and become
 CHAPTER 77  Canine and Feline Hepatozoonosis
FIGURE 77-1  Geographic distribution of reported Hepatozoon canis and Hepatozoon americanum infections. Both species exist in the United States. (Blue, Hepatozoon canis; red,
Hepatozoon americanum; purple both species.)
FIGURE 77-2  Hepatozoon americanum oocysts from the hemocoel of an Amblyomma
maculatum tick. Hundreds of small, round sporocysts are present within each oocyst. Each
sporocyst contains 10 to 26 infective sporozoites.
gamonts, which can then be ingested by feeding ticks (Figure
77-8). The larger macromerozoites are believed to be responsible
for the production of secondary meronts in the target tissues,
which continue the asexual cycle of merogony. Another tissue
stage found in dogs with H. canis but not H. americanum infec-
tions is a small monozoic cyst that resembles the cystozoite found
in transport hosts of other Hepatozoon species.39
749
Other modes of transmission also occur in hepatozoonosis.
Transplacental transmission from dam to puppies has been doc-
umented for H. canis infections and likely occurs in H. america-
num infections as well.40 Ingestion of tissue from wild animals
or prey may transmit H. americanum.41-43 Laboratory rodents
fed H. americanum oocysts developed cystozoites, but they
did not develop gamonts or meronts or become ill. Cystozoite-
laden rodent tissue fed to naïve dogs resulted in H. americanum
infection along with its classical clinical signs. Investigation of
a natural outbreak in a pack of hunting beagles revealed that
clinical signs of American canine hepatozoonosis arose 4 to
6 weeks after some of the dogs were allowed to consume a wild
rabbit carcass, whereas dogs not allowed to consume the rabbit
did not develop clinical signs. The predation route of transmis-
sion has not yet been proven to occur with H. canis.
The geographic distribution of American canine hepato-
zoonosis aligns closely with the range of the Gulf Coast tick.
Although this tick is endemic in the states that surround the Gulf
of Mexico, its range is expanding and it has been found as far
north as Kansas and Kentucky.44 Larval and nymphal stages of
A. maculatum preferentially feed on birds and small rodents.
Surveys of wildlife have revealed the presence of Hepatozoon
spp. that are related but not identical to H. americanum in small
rodents and rabbits.45 Although a reservoir species has not yet
been identified, dogs are likely an aberrant, rather than natural,
host of H. americanum. H. americanum or closely related species
have been identified in coyotes, bobcats, and ocelots.46-49 Because
these wild animals were in good physical condition at the time
of capture, they may represent a reservoir host in nature. In con-
trast to adult animals, coyote puppies developed classic signs of
H. americanum infection after experimental transmission.50
750 SECTION 4  Protozoal Diseases
FIGURE 77-3  Life cycle of Hepatozoon americanum.
FIGURE 77-4  Skeletal muscle containing a developing meront of H. americanum.
The mucopolysaccharide layers produced by the host cell protect the developing organ-
isms from the host’s immune system.
Dogs are believed to be the natural host reservoir of H. canis.
The brown dog tick, which serves as the definitive host of
H. canis, is found worldwide, and all three stages preferentially
feed on dogs. Life stages of H. canis or morphologically indis-
tinguishable Hepatozoon spp. have been reported in numerous
carnivore species around the world, including several species of
fox, jackal, African wild dog, hyena, palm civet, cheetah, leopard,
lion, and Pallas cat.30
FIGURE 77-5  Pyogranuloma in skeletal muscle of a dog infected with Hepatozoon
americanum. Zoites displace the nucleus in several of the inflammatory cells.
There is no sex or breed predilection for hepatozoonosis, but
hunting dogs, rural dogs, and dogs allowed to roam are most
at risk.
Clinical Features
Signs and Their Pathogenesis
Dogs with H. americanum infection exhibit a moderate to
severe illness, whereas most dogs infected with H. canis have no
or only mild signs of illness. Immunosuppression or concurrent
disease is an important factor in expression of illness in H. canis
but not in H. americanum infections. Co-infections with other
pathogens such as Ehrlichia, Babesia, Anaplasma, Leishmania,
parvovirus, or canine distemper virus can allow establishment
of a new H. canis infection as well as progression or reactivation
of an existing infection.
Clinical signs of H. americanum infection are associated
with the strong inflammatory response that occurs when
meronts rupture, leukocytes are recruited, and pyogranu-
lomas form in skeletal muscle. The earliest lesions occur
3 weeks after infection.51 As the organisms multiply, the
infection disseminates, which results in more severe inflam-
mation that waxes and wanes over time. The inflammation
causes fever and myositis, which is associated with locomo-
tor abnormalities and hyperesthesia (Table 77-2). The clini-
cal signs can mimic those of meningitis or discospondylitis.
Many affected dogs also develop bone lesions that are evi-
dent radiographically and resemble lesions seen in hypertro-
phic osteopathy, except they are proximal rather than distal
in distribution (Figure 77-9). It is unknown whether the bone
lesions result from nearby localized muscle inflammation or
from humoral factors. Dogs usually have a mucopurulent
ocular discharge that may be caused by pyogranulomatous
inflammation of the extraocular muscles or of the lacri-
mal gland (Figure 77-10). Return of ocular discharge is fre-
quently the first indication of a relapse following treatment.
Dogs with American canine hepatozoonosis frequently main-
tain a fairly normal appetite; however, weight loss, cachexia,
and muscle atrophy occur over time. There may be a history
of polyuria and polydipsia that is due to glomerulonephritis
or amyloidosis, which can occur secondary to long-standing
inflammation. Nephrotic syndrome and thromboembolic
complications may ensue. Less common clinical signs include
 CHAPTER 77  Canine and Feline Hepatozoonosis
FIGURE 77-6  Life cycle of Hepatozoon canis.
FIGURE 77-7  Hepatozoon canis meront in splenic tissue of a dog from Israel dem-
onstrating the typical “wheel spoke” pattern. (Courtesy of Dr. Gad Baneth, Koret School of
Veterinary Medicine, Israel.)
diarrhea, mucosal pallor, cough, abnormal lung sounds, and
lymphadenomegaly.
Most dogs infected with H. canis have a low level of parasit-
emia, with less than 5% of circulating leukocytes infected with
gamonts.52 These dogs have a limited inflammatory reaction.
About 15% of parasitemic dogs have a high level of parasitemia
(>800 gamonts/µL). The degree of parasitemia correlates with
the severity of clinical signs; the sickest dogs have a level of
parasitemia that approaches 100% of infected circulating leu-
kocytes. These dogs may have hepatitis, glomerulonephritis, or
pneumonitis in addition to severe anemia, fever, and cachexia.
Physical Examination Findings
With H. americanum infection, fever (up to 105.6°F or 40.9°C)
is common, although because of the waxing and waning nature
751
FIGURE 77-8  Two Hepatozoon canis gamonts in neutrophils on a blood smear. The
gamonts of Hepatozoon americanum are nearly identical in appearance to those of H. canis,
although they are rarely seen. 1000x magnification. (Courtesy of Dr. Gad Baneth, Koret
School of Veterinary Medicine, Israel.)
of the disease, body temperature may be normal at any given
time.23 Because of the myositis, dogs are typically evaluated for
gait abnormalities, which range from lameness or stiffness to
recumbency and an inability to rise. Hyperesthesia is common
and may appear as cervical, back, joint, or generalized pain.
Lethargy is common, and cachexia and generalized muscle atro-
phy may be apparent. Mucopurulent ocular discharge is often
present. Other ocular lesions seen on occasion include focal
retinal scars or hyper-reflectivity, increased retinal pigmenta-
tion, papilledema, and uveitis with active inflammatory fundic
lesions. Mucosal pallor and lymphadenomegaly may also be
present.
752 SECTION 4  Protozoal Diseases

TABLE 77-2
Frequency of Clinical Signs in 22 Dogs with American Canine
Hepatozoonosis
Clinical Sign Number of Dogs (%)
Fever 19 (86)
Weight loss 18 (82)
Mucopurulent ocular discharge 17 (77)
Low tear production 8 (36)
Muscle atrophy 14 (64)
Pain (all types) 14 (64)
Joints 2 (9)
Lumbar 4 (18)
Cervical 5 (23)
Generalized 3 (14)
Stiffness 12 (55)
Generalized weakness 9 (41)
Pelvic limb paresis and ataxia 5 (23)
Inability to rise 5 (23)
Anorexia 5 (23)
FIGURE 77-9  Radiograph of the pelvic limb of a dog with Hepatozoon americanum
From Vincent-Johnson NA. American canine hepatozoonosis. Vet Clin infection. There is smooth periosteal proliferation on the cranial aspect of the femur.
North Am Small Anim Pract. 2003;33:905-920. Data from Macintire (From Vincent-Johnson NA. American canine hepatozoonosis. Vet Clin North Am Small
DK, Vincent-Johnson N, Dillon AR, et al. Hepatozoonosis in dogs: 22 Anim Pract. 2003;33:905-920.)
cases (1989-1994). J Am Vet Med Assoc. 1997;210:916-922.

Dogs with mild H. canis infections may have pale mucous

membranes and lethargy. Dogs with high parasitemia often
exhibit fever, lethargy, and severe weight loss or cachexia, even if
they maintain a good appetite. Splenomegaly and lymphadeno-
megaly may be detected. Dogs often have physical examination
abnormalities that relate to the presence of co-infections or other
comorbidities.

Diagnosis
Laboratory Abnormalities
Complete Blood Count
The most common laboratory abnormality in H. americanum
infection is leukocytosis, which is often extreme (Table 77-3).
White cell counts are typically 20,000 to 200,000 cells/µL,
with reported means of 76,807 and 85,700 cells/µL.23,53 The
leukocytosis is due to a mature neutrophilia, although some-
times there is a mild to moderate left shift. A mild normocytic,
normochromic nonregenerative anemia is typical. Platelets are
usually normal to increased, sometimes with thrombocytosis
up to 916,000 platelets/µL. When thrombocytopenia is evident,
concurrent tick-borne diseases such as ehrlichiosis should be
considered. FIGURE 77-10  Rottweiler with Hepatozoon americanum infection. Note the hunched
The most common laboratory finding in H. canis infec- appearance, muscle atrophy, and mucopurulent ocular discharge.
tion is anemia, which is usually normocytic, normochromic,
and occasionally regenerative.52 Platelets are decreased about
one third of the time, but this may be due to concurrent infec- Serum Chemistry Profile
tion with Ehrlichia canis or other disease. The white blood cell In H. americanum infection, a mild elevation in the activity
count is typically normal in cases with low parasitemia but is of ALP is typical, possibly due to periosteal new bone forma-
elevated in dogs with high parasitemia. Some dogs have neu- tion. Serum glucose concentration is often decreased (40 to
trophil counts of 50,000 to 150,000/µL with close to 100% of 60 mg/dL) and occasionally as low as 5 mg/dL. This is not
these cells containing gamonts. a true hypoglycemia, but rather a laboratory artifact due to
 CHAPTER 77  Canine and Feline Hepatozoonosis 753

new bone formation occurs most frequently and severely on

TABLE 77-3 the diaphysis of long bones but occurs to a lesser degree on
Frequency of Laboratory and Radiographic Findings in 22 Dogs flat and irregular bones. The radiographic appearance can vary
from subtle irregular periosteal exostoses to thick parallel pseu-
with American Canine Hepatozoonosis
docortices. H. americanum infection typically affects the more
proximal bones while sparing the distal bones.56
Finding Number of Dogs (%)
In H. canis infections, radiographic bone lesions are rare.
Moderate to marked leukocytosis* 22 (100) Periostitis has only been described in three reports from Japan,
Mature neutrophilia 15 (68) India, and Italy, and in an experimental infection in Israel.4,57
Mild left shift 7 (31)
Elevated ALP activity 22 (100) Microbiologic Testing
Hypoglycemia 20 (91) Diagnostic assays currently available for hepatozoonosis in
dogs are described in Table 77-4.
Hypoalbuminemia 19 (86)
Periosteal proliferation on 18 (82) Cytologic Diagnosis
radiography The sexual stage of H. americanum and H. canis, the gamont,
Anemia (nonregenerative) 14 (64) appears as a light blue to clear oblong structure with a faintly
staining nucleus within the cytoplasm of neutrophils or monocytes
Hypocalcemia 14 (64)
stained with Diff Quik or Giemsa. The gamonts of the two species
Normal platelet count 11 (50) look nearly identical, although there is a slight difference in size:
Thrombocytosis 9 (41) H. canis gamonts measure approximately 11.0 × 4.3 µm while
Hyperphosphatemia 7 (32) H. americanum gamonts are slightly smaller at 8.8 × 3.9 µm.26
In dogs with H. americanum infection, gamonts are rarely
Low BUN concentration 7 (32)
found within monocytes or neutrophils on peripheral blood
Hyperglobulinemia 4 (11) smears. When present, infected cells rarely exceed 0.1% of the
Hypercalcemia 2 (9) circulating leukocytes; several thousand cells may have to be
Elevated CK activity 1 (5) examined before an infected cell is found. The chance that gam-
onts are detected can be increased by examination of buffy-coat
From Vincent-Johnson NA. American canine hepatozoonosis. Vet Clin smears. Neither bone marrow nor lymph node aspirates are of
North Am Small Anim Pract. 2003;33:905-920. Data from Macintire much value in making a definitive diagnosis of H. americanum
DK, Vincent-Johnson N, Dillon AR, et al. Hepatozoonosis in dogs: 22 infection; organisms are not usually seen in these specimens.
cases (1989-1994). J Am Vet Med Assoc. 1997;210:916-922. In most dogs with H. canis infections, gamonts are found
*Minimum white cell count was 27,800 cells/µL. on blood smear examination in 0.5% to 5% of neutrophils and
monocytes. Parasitemia may approach 100% in heavy infec-
tions. Gamonts may also be identified in dogs that are apparently
utilization of glucose in the specimen by the high number of healthy.
white blood cells. Hypoalbuminemia is common and may be
due to chronic inflammation, decreased protein intake, or Serologic Diagnosis
renal loss from secondary glomerulonephritis or amyloidosis. An ELISA assay for detection of antibodies to H. americanum
Except in dogs with significant renal damage, BUN concentra- has been developed at Oklahoma State University for research
tion is often low. The combination of increased activity of purposes, but this assay is not available commercially to veteri-
serum ALP and low glucose, BUN, and albumin concentra- nary practitioners.58 Both an indirect immunofluorescent anti-
tions may mislead the clinician to suspect liver disease. Serum body (IFA) assay and ELISA assays for H. canis antibodies have
bile acid concentrations are normal or only slightly elevated. been developed and used for epidemiologic studies in countries
Surprisingly, the activity of serum CK is consistently within outside of the United States.59-62 Positive serologic test results
the normal range even though H. americanum infection causes indicate exposure but do not prove active infection or correlate
severe myositis. with clinical disease. Thus, serologic testing is mostly of use for
Common serum chemistry findings in H. canis infection epidemiologic studies and for screening blood donor animals.
include hyperproteinemia, hyperglobulinemia, and hypoalbu-
minemia. The hyperglobulinemia is due to a polyclonal gam- Molecular Diagnosis Using the Polymerase Chain Reaction
mopathy.52,54 Increases in the activities of ALP and CK are A real-time PCR assay that detects Hepatozoon spp. is available
usually present. commercially to veterinary practitioners through the Auburn
University Molecular Diagnostics Laboratory.63 Other com-
Diagnostic Imaging mercial veterinary diagnostic laboratories also offer real-time
Plain Radiography PCR assays for detection of Hepatozoon species. The assay per-
Many dogs with H. americanum infection show radiographic formed at Auburn University detects as few as seven genomic
abnormalities of the skeletal system; therefore, radiographs of copies of Hepatozoon spp. per mL of blood, and it distinguishes
the pelvis or long bones can be useful to support a clinical diag- between infection with H. canis and H. americanum.16,17
nosis of American canine hepatozoonosis. Young dogs are more Although highly sensitive and specific, false negatives can occur
likely than older dogs to show bone lesions. In experimental early in the course of infection or in dogs with chronic disease
infections, bone lesions are recognizable histologically within where the numbers of circulating gamonts are extremely low or
5 weeks postexposure.55 Disseminated, symmetric periosteal absent. Muscle biopsy should be performed in dogs suspected
754 SECTION 4  Protozoal Diseases
TABLE 77-4
Diagnostic Assays Currently Available for Hepatozoonosis in Dogs
Assay Specimen Type Target
Cytologic examination Whole blood smears Hepatozoon spp.
gamonts in leukocytes
Antibody serology Serum Antibodies to H. canis
(ELISA or immuno­
fluorescent antibody)
Real-time PCR assays Whole blood H. americanum or
H. canis DNA
Histopathology Muscle biopsy H. americanum cysts,
specimens meronts, and pyogran- infection, but requires anesthesia.
ulomas
to have H. americanum infection but that have negative PCR
assay results.
Pathologic Findings
Gross Pathologic Findings
In dogs with H. americanum infection, muscle atrophy and
cachexia are consistent findings on gross necropsy examination.
Pyogranulomas may appear as multiple 1- to 2-mm–diameter
white-tan foci.23 These are scattered diffusely throughout skeletal
and cardiac muscle but may also be observed in other tissues.
Roughening and thickening of bony surfaces are often present.
Less common findings include lymphadenomegaly, congestion
of the gastric mucosa, splenic coagulative necrosis, and pulmonary
congestion.
In dogs with H. canis infection, the spectrum of gross patho-
logic lesions depends on the severity of the infection. In heavy
infections, splenomegaly and hepatomegaly are common findings
and these organs often exhibit diffuse white necrotic foci that are
1 to 2 mm in diameter.14 The necrotic areas may be larger and
nodular and can also be found in other tissues, such as pancreas
and pleura. Lymphadenomegaly is common, and pneumonia
may be present.
Histopathologic Findings
For H. americanum infections, muscle biopsy has long been con-
sidered the most reliable method for diagnosis. Small pieces of
muscle (approximately 2 cm × 2 cm) are obtained from the biceps
femoris or semitendinosus muscles under general anesthesia.25
Submission of multiple specimens increases sensitivity, especially
in low-level infections. Muscle lesions consist of large onion-skin
cysts, pyogranulomas, and myositis. The cysts are round to oval
and have mean cross-section dimensions of 186 × 150 µm.26 At
the center of the cyst is a host cell nucleus and protozoa, but
depending on the plane of the cut surface, these may or may not
be visible. Surrounding the center of the cyst are concentric layers
of mucopolysaccharide. Occasionally meronts in various stages
of development are observed within these cystic structures. The
highly vascular pyogranulomas are packed with neutrophils and
Performance
False negatives are common because of low circulat-
ing numbers of gamonts, especially in clinical
H. americanum infections.
Positive test results indicate exposure but do not
indicate the presence of the organism in circulat-
ing leukocytes and do not correlate with clinical
disease.
Sensitivity and specificity may vary depending on
assay design, but in general PCR assays are more
sensitive than cytology. False-negative results may
occur when organism levels are very low, such as
early in disease or in very chronic infections.
Most reliable method of diagnosis of H. americanum
macrophages, many of which contain a single zoite. Myositis is
characterized by muscle necrosis and atrophy with infiltration of
neutrophils, macrophages, and occasional lymphocytes between
muscle fibers. Although consistently identified in skeletal and car-
diac muscle, H. americanum cysts, meronts, and pyogranulomas
can sporadically be found in adipose tissue, intestinal smooth
muscle, pancreas, liver, lymph node, spleen, skin, salivary gland,
lung, and kidney.64 Renal damage is common and manifests as
focal pyogranulomatous inflammation with mild glomerulone-
phritis, lymphoplasmacytic interstitial nephritis, mesangioprolif-
erative glomerulonephritis, or occasionally amyloidosis. Amyloid
deposits have also been found in the spleen, lymph nodes, small
intestines, and liver of affected dogs. Various organs can show
vascular changes, which include fibrinoid degeneration of ves-
sel walls, mineralization and proliferation of the vascular intima,
and pyogranulomatous vasculitis.
In H. canis infections, “wheel-spoked” meronts are present in
varying numbers and stages of development throughout hemo-
lymphatic target organs, such as bone marrow, spleen, and
lymph nodes. The meronts are approximately 30 µm in diameter
and on central cross-section appear as a circle of micromerozoite
nuclei surrounding a central core. Meronts can be found in small
numbers as an incidental finding in dogs from endemic areas.65
Inflammation associated with meronts can range from none to
very severe. In heavy infections, microscopic lesions may include
hepatitis with Kupffer’s cell hyperplasia and inflammatory infil-
trates, interstitial pneumonia and thickening of alveolar septae
with inflammatory infiltrates, glomerulonephritis or interstitial
nephritis with multifocal necrosis, and focal splenic necrosis.
Treatment and Prognosis
Although no treatment effectively eliminates the tissue stages
of H. americanum, remission of clinical signs can be achieved
using a combination of a trimethoprim-sulfa, clindamycin,
and pyrimethamine for 14 days (Table 77-5).66 Response to
therapy is usually dramatic, with resolution of clinical abnor-
malities within 48 to 72 hours of initiating therapy. Ponazuril
 CHAPTER 77  Canine and Feline Hepatozoonosis 755

TABLE 77-5
Antimicrobial Drugs Indicated for Treatment of American Canine Hepatozoonosis

Drug Dose* (mg/kg) Route Interval (hours) Duration (days)

TCP combination:
Trimethoprim-sulfonamide and 15 PO 12 14
Clindamycin and 10 PO 8 14
Pyrimethamine and 0.25 PO 24 14
Decoquinate† 10-20 PO 12 730

Adapted from Baneth G, Macintire DK, Vincent-Johnson N, et al. Hepatozoonosis. In Greene CE, ed. Infectious Diseases of the Dog and Cat, 3 ed.
St. Louis, MO: Saunders; 2006:698-711.
TCP, Trimethoprim-sulfadiazine, clindamycin, and pyrimethamine; PO, by mouth.
*Dose per administration at specified interval.
†Use the decoquinate 6% (27.2 g/lb) powder, Deccox (Alpharma Inc., Fort Lee, NJ). One teaspoonful is equivalent to approximately 180 mg

decoquinate. Administer at a rate of 1 teaspoon per 10 kg of body weight and feed q12h mixed into moist dog food.

(10 mg/kg PO q12h for 14 days) is an alternative therapeutic
option.67 Either of these protocols must be followed by long-
term administration of decoquinate in order to prevent relapse.
Without the addition of decoquinate, relapse occurs in most
dogs 2 to 6 months following treatment. Although these dogs
generally respond well to another round of combination ther-
apy, subsequent relapses occur more frequently, and eventually
disease becomes refractory to treatment. Persistent infections
and multiple relapses lead to complications such as glomeru-
lonephropathy, amyloidosis, vasculitis, and cachexia, which
carry a guarded to poor prognosis. Decoquinate, a livestock
anticoccidial agent, apparently breaks the asexual recycling
of infection by arresting development of the merozoites after
their release from meronts. Two years of daily decoquinate
therapy is recommended.66 Alternatively, a PCR assay can be
performed every 3 to 6 months, and when the PCR becomes
negative, treatment can be discontinued. Mild relapses can
occur even during treatment with decoquinate. If clinical signs
are severe, another course of combination therapy is indicated.
Long-term administration of decoquinate results in extended
survival times and excellent quality of life with a good prog-
nosis. During the initial few days of treatment with either
antiprotozoal regimen, administration of a nonsteroidal anti-
inflammatory drug at standard dosages can provide relief from
fever and pain.
Hepatozoon canis infections are treated with imidocarb
dipropionate (5 to 6 mg/kg SC or IM every 14 days) until
gamonts are no longer seen on blood smear examination.52
Although some dogs require only one or two treatments, at
least 8 weeks of treatment may be required for dogs with heavy
infections. Doxycycline (10 mg/kg/day PO for 21 to 28 days) is
frequently given in conjunction with imidocarb dipropionate.
It has been recommended that all dogs, even those with only
mild disease, be treated, because parasitemia may increase over
time. A recent study showed that despite negative blood smears,
dogs remained positive by PCR or buffy-coat examination even
when treated repeatedly over 8 months.68 This study also found
that buffy-coat examination was twice as sensitive as blood
smear examination in detecting gamonts, and PCR was even
more sensitive for detection of persistent infection. Addition of
toltrazuril does not seem to provide any additional benefit to
imidocarb therapy.69 Despite lack of a parasitologic cure, clini-
cal cure is achieved in many dogs. Dogs with low parasitemia
generally have a good long-term prognosis for survival, whereas
those with high parasitemia have a guarded prognosis.
Immunity and Vaccination
Current treatments do not cure H. americanum or H. canis
infections, so dogs remain susceptible to relapse of disease, espe-
cially if immunosuppressed. In experimental H. canis infections,
treatment with an immunosuppressive dose of glucocorticoids
was followed by the appearance of parasitemia.33 Vaccines for
H. americanum or H. canis infections are not available.
Prevention
Since both H. canis and H. americanum are transmitted by
ingestion of ticks, the use of topical acaricides, environmental
parasiticides, and immediate removal of ticks from dogs are the
most important means of prevention. Because H. americanum,
and possibly H. canis, can also be transmitted through ingestion
of cystozoites in muscle from other animals, dogs should not be
allowed to roam, scavenge, or engage in predatory behavior.
Dogs should not be fed uncooked or undercooked game meat.
Although Hepatozoon spp. have not been shown to be transmitted
through blood transfusion, it seems logical that dogs infected
with Hepatozoon species be avoided as blood donor dogs. To
prevent congenital infections of puppies, infected females either
should not be bred or should receive treatment before breeding.
Public Health Aspects
Only one case of human infection with a Hepatozoon spp. has
been reported. Gamonts were identified on blood smears of
an adult man from the Philippines who suffered from anemia
and icterus, but no parasites were found in liver or bone mar-
row biopsies.70 Because hepatozoonosis is transmitted by tick
ingestion rather than a tick bite, tick ingestion is an unlikely
route of transmission for humans. However, caution should
be used when removing ticks from dogs or when handling
tick-infested dogs or blood from infected dogs because of the
756 SECTION 4  Protozoal Diseases
unknown zoonotic potential of organisms that cause canine
disease and the possibility that known human pathogens may
also be present.
Hepatozoonosis in Domestic Cats
Feline hepatozoonosis occurs primarily in areas where canine
hepatozoonosis exists, and it has been reported in a number
of countries, including India, South Africa, Nigeria, Brazil,
Israel, Spain, France, and Thailand.30,31 In the United States,
there is one report of hepatozoonosis in a domestic cat from
Hawaii.71 A survey of blood specimens from stray cats in
Bangkok, Thailand, showed that 32.3% were positive by
PCR for a Hepatozoon sp. most closely related to H. canis,
whereas only 0.7% of the cats had gamonts observed on blood
CASE EXAMPLE
Signalment: “Pepper,” a 1.5-year-old male miniature schnauzer
from Opelika, AL (Figure 77-11)
History: Pepper was seen for a 1-month duration of illness
characterized by intermittent fever (104.0°F to 105.0°F [40.0°C
to 40.6°C]), lethargy, and ocular discharge. His owner reported
that Pepper had also been stiff and reluctant to move. Despite
a fairly good appetite, he had lost weight and muscle mass.
The fever and other clinical signs had been nonresponsive
to antibiotics, which included enrofloxacin and doxycycline,
prescribed at the onset of illness. Pepper was a house dog
but spent time outdoors in a rural environment. No vomiting,
coughing, or sneezing had been reported, but he had had
mild diarrhea at the onset of illness that resolved after a week.
He had also had a mild increase in thirst and urination. He was
fed a high-quality commercial dry dog food, received monthly
heartworm preventive, and was current on all vaccinations,
including canine distemper, hepatitis, parvovirus, and rabies
vaccines. He had not traveled outside of the local area. The
owner mentioned that she had had another dog that died
about 1 year earlier after exhibiting similar clinical signs, but a
diagnosis was not made.
Current medications: Aspirin 11 mg/kg PO q12h for pain.
Physical Examination:
Body Weight: 7.6 kg.
General: Quiet, slightly lethargic, but responsive. Reluctant to
walk or stand; appeared painful. T = 103.8°F (39.9°C), HR = 96
beats/min, RR = 32 breaths/min, mucous membranes pink,
CRT = 1.5 s.
Integument: The skin and haircoat appeared normal with no
evidence of ectoparasites.
Eyes, Ears, Nose, and Mouth: A moderate amount of
mucopurulent ocular discharge was present bilaterally. No
other abnormalities were identified.
Musculoskeletal: Body condition score was 3/9 with
moderate generalized muscle atrophy. The dog exhibited
hyperesthesia during palpation of trunk, head, neck, and
limb muscles. The dog’s gait was very stiff and slow.
All Other Systems: Apart from abdominal splinting on
palpation of the abdomen, no clinically significant
abnormalities were present.
smears.7 In Israel, Hepatozoon DNA was amplified from the
blood of 36% of 152 cats, and infected cats were significantly
more likely to have access to the outdoors.31 The vast majority
of cats were infected with an organism that resembled H. felis,
although 2 cats were infected with an organism that resembled
H. canis. Most infections were subclinical. Meronts have also
been found in the myocardium and skeletal muscles of cats
with no signs of illness. Cats with clinical hepatozoonosis are
commonly immunosuppressed because of infection with FIV
or FeLV. A range of clinical signs has been reported, including
weakness, intermittent anorexia, weight loss, fever, lymph-
adenomegaly, ulcerative glossitis, hypersalivation, anemia,
serous ocular discharge, and icterus. Treatment success has
been reported with oral doxycycline at 5 mg/kg PO once a day
for 10 days.72
FIGURE 77-11  “Pepper,” a 1.5-year-old male miniature schnauzer with H.
americanum infection.
Ophthalmic Exam: Pupillary light reflex (direct and
consensual): Normal.
Conjunctiva: injected bilaterally, mucopurulent discharge bilat-
erally.
Cornea and lens: Normal.
Schirmer Tear Test: Right eye = 18 mm; left eye = 16 mm (normal
> 15 mm).
Fluorescein stain: Negative.
Tonometry: Normal.
Fundoscopic examination: A small grayish, slightly raised focal
lesion was identified in the right fundus.
Laboratory Findings:
CBC:
PCV 38.7% (37%-55%)
MCV 68.7 fL (60-77 fL)
MCHC 35.4 g/dL (32-36 g/dL)
WBC 39,200 cells/µL (6000-17,000 cells/µL)
Neutrophils 35,672 cells/µL (3000-11,400 cells/µL)
Lymphocytes 1176 cells/µL (1000-4000 cells/µL)
Monocytes 2352 cells/µL (150-1200 cells/µL)
Eosinophils 0 (100-750 cells/µL)
Platelets 607,000 platelets/µL (200,000-400,000 platelets/µL).
The clinical pathologist’s blood smear review showed no
evidence of microorganisms.
 CHAPTER 77  Canine and Feline Hepatozoonosis
Serum Chemistry Profile:
BUN 8 mg/dL (10-25 mg/dL)
Creatinine 0.6 mg/dL (0.3-1.0 mg/dL)
ALP 142 U/L (19-50 U/L)
ALT 37 U/L (17-66 U/L)
Creatine kinase 148 U/L (92-357 U/L)
Total bilirubin 0.3 mg/dL (0.1-0.3 mg/dL)
Glucose 52 mg/dL (80-100 mg/dL)
Sodium 153 mmol/L (146-160 mmol/L)
Potassium 4.7 mmol/L (3.5-5.9 mmol/L)
Chloride 118 mmol/L (108-125 mmol/L)
Calcium 10.3 mg/dL (9.5-11.8 mg/dL)
Phosphorus 3.9 mg/dL (3.3-5.8 mg/dL)
Total protein 6.4 g/dL (5.1-7.3 g/dL)
Albumin 2.7 g/dL (2.6-3.5 g/dL)
Globulin 3.7 g/dL (3.6-5.0 g/dL)
Total CO2 21.8 mmol/L (13.9-31.5 mmol/L).
Urinalysis: SGr 1.035; pH 8.0, protein 1+, bilirubin negative,
hemoprotein negative, glucose negative, 0 WBC/HPF, 0 RBC/
HPF, no crystals, moderate lipid droplets.
Imaging Findings:
Pelvic Radiographs: Bilateral smooth periosteal bone
formation was present on the ilium and femurs.
Abdominal Ultrasound Examination: No abnormalities were
detected.
Bone Marrow Aspirate: The bone marrow smears were
highly cellular with many particles. Megakaryocytes were
adequate. In a 500-cell count, the M:E ratio was 1.7 (reference
range 1.3-2.1) with normal maturation sequences in both
red and white series. Multiple smears examined showed no
evidence of microorganisms.
Muscle Biopsy: Histopathology showed infiltration of
neutrophils and monocytes between muscle fibers. There
were rare onion-skin cysts and one focal pyogranuloma.
Many of the monocytes and neutrophils within the
granuloma contained a single zoite. Findings were consistent
with Hepatozoon americanum infection.
Cytology Findings: Although negative at the initial
evaluation, blood smears and buffy-coat smears performed
a few days later showed oblong “jellybean” inclusions in
SUGGESTED READINGS
Baneth G. Perspectives on canine and feline hepatozoonosis. Vet Para-
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Little SE, Allen KE, Johnson EM, et  al. New developments in canine
hepatozoonosis in North America: a review. 4th International Canine
Vector-Borne Disease Symposium, Seville, Spain, 26-28 March 2009.
Parasites Vectors. 2009;2(Suppl I):S5.
Vincent-Johnson NA. American canine hepatozoonosis. Vet Clin North
Am Small Anim Pract. 2003;33:905-920.
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Diagnosis: American canine hepatozoonosis (Hepatozoon
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Treatment: Triple combination therapy consisting of
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29. Little L, Baneth G. Cutaneous Hepatozoon canis infection in a dog
from New Jersey. J Vet Diagn Invest. 2011;23:585-588.
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Philadelphia, PA: WB Saunders; 2006:698-711.
31. Baneth G, Sheiner A, Eyal O, et al. Hepatozoon felis infection in
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32. Mathew JS, Ewing SA, Panciera RJ, et al. Experimental transmis-
sion of Hepatozoon americanum to dogs by the Gulf Coast tick,
Amblyomma maculatum Koch. J Parasitol. 1998;80:1-14.
33. Baneth G, Samish M, Alekseev E, et  al. Transmission of Hepa-
tozoon canis to dogs by naturally fed or percutaneously injected
Rhipicephalus sanguineus ticks. J Parasitol. 2001;87:606-611.
34. Murata T, Inoue M, Taura Y, et al. Detection of Hepatozoon canis
oocyst from ticks collected from the infected dogs. J Vet Med Sci.
1995;57:111-112.
35. Forlano M, Scofield A, Elisei C, et  al. Diagnosis of Hepatozoon
spp. in Amblyomma ovale and its experimental transmission in
domestic dogs in Brazil. Vet Parasitol. 2005;134:1-7.
36. de Miranda RL, de Castro JR, Olegario MM, et al. Oocysts of Hep-
atozoon canis in Rhipicephalus (Boophilus) microplus collected
from a naturally infected dog. Vet Parasitol. 2011;177:392-396.
37. Ewing SA, DuBois JG, Mathew JS, et al. Larval Gulf Coast ticks
(Amblyomma maculatum) [Acari: Ixodidae] as host for Hepa-
tozoon americanum [Apicomplexa: Adeleorina]. Vet Parasitol.
2002;103:43-51.
38. Baneth G, Samish M, Shkap V. Life cycle of Hepatozoon canis
(Apicomplexa: Adeleorina: hepatozoidae) in the tick Rhipicepha-
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39. Baneth G, Shkap V. Monozoic cysts of Hepatozoon canis. J Parasi-
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40. Murata T, Makoto I, Susumu T, et  al. Vertical transmission of
Hepatozoon canis in dogs. J Vet Med Sci. 1993;55:867-868.
41. Johnson EM, Allen KE, Panciera RJ, et  al. Infectivity of Hep-
atozoon americanum cystozoites for a dog. Vet Parasitol.
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42. Johnson EM, Allen KE, Panciera RJ, et al. Experimental transmis-
sion of Hepatozoon americanum to New Zealand White rabbits
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43. Johnson EM, Panciera RJ, Allen KE, et  al. Alternate pathway of
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44. Ewing SA, Mathew JS, Panciera RJ. Transmission of Hepatozoon
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49. Kocan AA, Breshears M, Cummings C, et  al. Naturally occur-
ring hepatozoonosis in coyotes from Oklahoma. J Wildl Dis.
1999;35(1):86-89.
50. Kocan AA, Cummings CA, Panciera RJ, et al. Naturally occurring
and experimentally transmitted Hepatozoon americanum in coy-
otes from Oklahoma. J Wildl Dis. 2000;36(1):149-153.
51. Panciera RJ, Ewing SA, Mathew JS, et  al. Canine hepatozoono-
sis: comparison of lesions and parasites in skeletal muscle of dogs
experimentally or naturally infected with Hepatozoon america-
num. Vet Parasitol. 1999;82:261-272.
52. Baneth G, Weigler B. Retrospective case-control study of hepatozo-
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retrospective study of 15 natural occurring cases. J Am Anim Hosp
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54. Gavazza A, Bizzeti M, Papini R. Observations on dogs found nat-
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55. Drost WT, Cummings CA, Mathew JS, et  al. Determination of
time of onset and location of early skeletal lesions in young dogs
experimentally infected with Hepatozoon americanum using bone
scintigraphy. Vet Radiol Ultrasound. 2003;44:86-91.
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 CHAPTER 77  Canine and Feline Hepatozoonosis
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66. Macintire DK, Vincent-Johnson NA, Kane CW, et  al. Treatment
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domestic cat. Feline Pract. 1995;23:10-12.
CHAPTER 78
Trypanosomiasis
Stephen C. Barr, Ashley B. Saunders, and Jane E. Sykes
Overview of American Trypanosomiasis
First Described: Chagas’ disease (American trypanosomiasis)
was first described in humans in 1909 (Carlos Chagas).1
Cause: Trypanosoma cruzi, a protozoan parasite (phylum
Sarcomastigophora, family Trypanosomatidae)
Affected Hosts: Dogs, humans, and more than 150 other
domestic or wildlife mammalian species
Geographic Distribution: Central and South America; to a
lesser extent southern United States (especially south-
eastern Texas)
Mode of Transmission: A bite from, or ingestion of, reduviid
(kissing bug) vectors, primarily Triatoma species. Trans-
mission through blood transfusion and vertical transmis-
sion can occur.
Major Clinical Signs: American trypanosomiasis in pup-
pies manifests as lethargy, inappetence, mucosal pallor,
lymphadenopathy, splenomegaly, arrhythmias, or sud-
den death. Neurologic signs such as pelvic limb ataxia
and exaggerated spinal reflexes may also occur. Adult
dogs may show lethargy, arrhythmias, signs of right- or
left-sided congestive heart failure, or sudden death.
Differential Diagnoses: The differential diagnoses for sus-
pected Chagas’ myocarditis include dilated cardiomy-
opathy of large-breed dogs, parvoviral myocarditis in
puppies, canine monocytic ehrlichiosis, babesiosis, and
leishmaniosis. The primary differential diagnoses for
suspected Chagas’ meningoencephalitis in puppies are
canine distemper virus infection and neosporosis, but
other infectious and inflammatory causes of meningoen-
cephalitis should also be considered.
Human Health Significance: Transmission to humans has the
potential to occur after needle-stick injuries; caution is
advised when handling tissues or clinical specimens from
dogs with suspected trypanosomiasis.
AMERICAN TRYPANOSOMIASIS
Etiology and Epidemiology
Chagas’ disease, or American trypanosomiasis, is caused by the
flagellated protozoan Trypanosoma cruzi. In North America,
disease in dogs usually manifests as cardiac disease typified
by arrhythmias or myocarditis (acute or chronic), and, rarely,
760
neurologic disease.2-7 However, many infected dogs are sub-
clinically infected for life. Cats can become infected, but dis-
ease in cats has not been recognized. Trypanosoma cruzi is an
important vector-borne zoonosis in the Americas, particularly
South and parts of Central America, and is the leading cause of
dilated cardiomyopathy in humans.8 Within the United States,
most cases in dogs occur in Texas and especially involve work-
ing dogs that reside in southeastern Texas (Figure 78-1).9-11
A few affected dogs have been reported from other southern
states,5,6,12-14 and as far north as Missouri.15 The seroprevalence
in dogs from Louisiana and Texas has generally been in the
range of 12% to 22%, with seroprevalences of <10% in other
states where canine disease has been identified.16 Canine Cha-
gas’ disease is of importance to veterinary practitioners because
it can be difficult to diagnose, is a potential zoonosis, and there
is a lack of therapeutic options.
Trypanosoma cruzi exists in three morphologic forms. The
form that circulates freely in the host’s peripheral blood is the
trypomastigote. This form is 15 to 20 µm long, with a flattened
spindle-shaped body and a centrally placed vesicular nucleus.
A single flagellum originates near a large, subterminal kineto-
plast (situated posterior to the nucleus) and passes along the
body to project anteriorly (Figure 78-2). The host intracellular
or amastigote form is approximately 1.5 to 4.0 µm in diameter,
is roughly spheroid, and contains both a nucleus and a rod-like
kinetoplast. A small flagellum is present, but this is rarely obvi-
ous under light microscopy. The third morphologic form, the
epimastigote, is found in the arthropod vector, a reduviid or
“kissing bug” (subfamily Triatomae). Epimastigotes are flagel-
lated and spindle shaped with the kinetoplast situated anterior to
the nucleus. When a vector is involved in transmission, infection
occurs when trypomastigotes are deposited in the triatomine’s
feces at the insect bite site. This is the main mode of transmission
to humans in South America.
Transmission of T. cruzi in endemic countries depends
on the confluence of vectors, reservoirs, parasites, and hosts
(both people and animals) in a single habitat. Only three Tri-
atomae species (Triatoma infestans [the main vector in South
America], Triatoma dimidiata, and Rhodnius prolixus) that
feed on man in endemic regions in South America display the
appropriate behavior that enables them to transmit T. cruzi
effectively. These species feed on blood from both people and
domestic reservoir mammals (dog, cat, guinea pigs), reproduce
prolifically while cohabiting close to people, and defecate soon
after taking a blood meal, meaning that they are usually still
on the host near the bite wound when they defecate.17 Infec-
tion rates in these vectors can be as high as 100% south of the
equator. By contrast, domestic transmission cycles probably do
not occur in the United States, except in areas of southeastern

A Maryland and in most other southern states (Texas, Louisiana,
B Alternatively, they spread hematogenously from the site of
FIGURE 78-1  A, Approximate distribution of Chagas’ disease in humans in the
Americas. Insect-transmitted human Chagas’ disease has also been rarely reported in Ten-
nessee, California and Louisiana. B, States within the United States where Chagas’ disease
is most prevalent in dogs. Disease in Texas primarily occurs in the southeast. Occasional
cases have been reported in other southern states and as far north as Virginia and Missouri.
Texas where there is evidence to suggest that the dog can be
involved in domestic transmission cycles involving vectors and
man.18 Generally, though, the two principal vectors in the
United States (Triatoma protracta and Triatoma sanguisuga)
have low infection rates (20%), display different feeding hab-
its, and defecate about 20 minutes after feeding, often when
they have long fled the host.19 As a result, it is more likely that
dogs in North America become infected when they eat infected
triatomines or their excreta. Certainly, opossums20 and arma-
dillos21 become infected by this route, and outbreaks have
occurred in humans following ingestion of contaminated food
CHAPTER 78  Trypanosomiasis 761
FIGURE 78-2  Trypomastigotes of Trypanosoma cruzi in a blood smear of a dog.
Wright-Giemsa stain, 1000× oil magnification. (From Barr SC. Canine Chagas’ disease
(American trypanosomiasis) in North America. Vet Clin North Am. 2009;39:1055-1064.)
or drinks.22 Blood transfusion and transplacental transmission
can also occur, and infection after ingestion of infected milk
from lactating bitches has been proposed.6 Whether ingestion
of reservoir hosts leads to transmission is unclear.
Within the United States, the principal wildlife reservoir
hosts of T. cruzi in the eastern seaboard states south from
and Oklahoma, to name a few) are opossums and raccoons.
Armadillos can also be infected.23 Various mouse, squirrel, and
rat species are the main reservoir hosts in New Mexico and Cal-
ifornia.24 Isolates of T. cruzi from vectors and animal reservoirs
in North America are less pathogenic in mice than South Ameri-
can isolates.23,25 Experimental inoculation of T. cruzi isolates
from opossums and armadillos into dogs produces a disease
consistent with naturally acquired acute and chronic canine try-
panosomiasis, so it is likely that dogs in nature are infected with
the same isolates as these wildlife hosts.2-6 Six genotypes of T.
cruzi have been identified (TcI through TcVI, also known as
discrete typing units or DTUs), which appear to differ in their
vector preferences and the extent to which they cause disease.26
After infection, trypomastigotes enter macrophages, transform
into amastigotes, and multiply by binary fission (Figure 78-3).
infection, infect myocardiocytes, transform into amastigotes,
multiply, and transform back into trypomastigotes. The host
cell then ruptures and trypomastigotes are released back into
circulation. Parasitemia in dogs appears as early as 3 days
postinfection (DPI), peaks at 17 DPI, and is cytologically
undetectable (subpatent infection) by 30 DPI.2 Clinical signs
of acute myocarditis, should they occur, develop about 14 DPI
with recovery occurring around 28 DPI.2 Rapid intracellular
multiplication ensures a rapid rise in parasitemia before effec-
tive immunity develops. Parasitemia steadily rises as more and
more intracellular multiplication cycles add to the number
of circulating trypomastigotes. The vector becomes infected
when it ingests circulating trypomastigotes, which transform
into epimastigotes and multiply by binary fission. Transforma-
tion of the epimastigotes back into trypomastigotes occurs in
the vector’s hindgut before the trypomastigotes are passed in
the feces.
762 SECTION 4  Protozoal Diseases
FIGURE 78-3  Life cycle of Trypanosoma cruzi. Dogs are infected when a triatome
(reduviid bug) defecates at its bite site, and possibly when dogs ingest infected triatomes.
Other routes of transmission may also predominate in the United States (see text). Trypo-
mastigotes infect cardiomyocytes (as well as other tissues) and form amastigotes, which
replicate locally, leading to myocarditis.
Clinical Features
Signs and Their Pathogenesis
As in humans, there are three phases of Chagas’ myocarditis in
dogs: acute, indeterminate (or latent), and chronic.2-6 Acute myo-
carditis results from cell damage and inflammation as trypomasti-
gotes rupture from myocardiocytes. Lethargy, fever, inappetence,
generalized lymphadenopathy, slow capillary refill time with
pale mucus membranes, and, in some cases, splenomegaly and
hepatomegaly are the main signs in puppies. Diarrhea can also
occur. In dogs over 6 months of age, parasitemia develops more
slowly and clinical signs are often much less severe or not appar-
ent at all. Sudden death, presumably from cardiac muscle failure
or conduction system failures leading to malignant arrhythmias,
is not a common occurrence. Although less common than signs
referable to cardiac abnormalities, neurologic signs referable to
meningoencephalitis (as a direct result of parasitic invasion of the
neurologic system) may also occur and include weakness, pelvic
limb ataxia, and exaggerated spinal reflexes suggestive of distem-
per. Humans with Chagas’ disease can also develop severe mega-
esophagus or megacolon due to parasympathetic denervation,22
but these have not been described in dogs.
Dogs that survive the acute phase enter a prolonged inde-
terminate phase typified by a lack of clinical signs. Parasitemia
becomes cytologically undetectable at about 30 DPI, although
it can still be demonstrated by blood culture. The ECG is usu-
ally normal during this phase, although ventricular arrhythmias
can be induced by exercise.3 Although not all dogs progress to
develop chronic disease, some develop chronic myocarditis with
cardiac dilatation over the subsequent 8 to 36 months.2,3 ECG
abnormalities become more prevalent in these dogs and may
even result in sudden death. Clinical signs referable to right-sided
and eventually, in some, left-sided heart failure occur and can
include exercise intolerance, pulse deficits, ascites, pleural effu-
sion, hepatomegaly, and jugular venous congestion.2 Chronic
Chagas’ myocarditis is clinically indistinguishable from dilated
cardiomyopathy of large breed dogs.4-6 The pathogenesis of the
cardiomyopathy is unknown, but proposed mechanisms include
damage to myocardiocytes or the autonomic nervous system by
immune-mediated mechanisms or toxic parasitic products, and/or
microvascular disease coupled with platelet dysfunction.27,28
Cardiac dilatation occurs when fibrosis no longer permits effi-
cient compensatory hypertrophy.27,29 Some T. cruzi isolates that
infect dogs in the United States are not pathogenic but produce
a marked serologic response and a low-level parasitemia fol-
lowing immunosuppression.2,30 Reactivation of infection with
immunosuppression can occur in human patients.22
Physical Examination Findings
The most common physical examination findings in puppies with
Chagas’ disease are lethargy, generalized lymphadenomegaly,
splenomegaly, pallor, and arrhythmias. Generalized subcutaneous
edema has also been reported.31 Dogs with chronic Chagas’ disease
may show signs of right- and/or left-sided congestive heart failure,
with tachypnea, ascites, or jugular pulses. Cardiac murmurs and/
or arrhythmias may be detected on auscultation of the heart.
Diagnosis
The key to diagnosis of Chagas’ disease is a high degree of clini-
cal suspicion. Chagas’ disease should be considered in any dog
with signs of myocarditis or cardiomyopathy, particularly if it
lives or has lived at any time—even years before evaluation—in
an endemic region.
Laboratory Abnormalities
Complete Blood Count
During the acute T. cruzi infection, the CBC of some dogs may
reveal anemia, leukocytosis due to a neutrophilia, eosinophilia,
monocytosis, and lymphocytosis.32,33 Thrombocytopenia, leu-
kopenia, and/or lymphopenia have also been documented.16
Dogs in the chronic phase of infection may have no hematologic
abnormalities.
Serum Biochemical Tests
Serum ALT activity, AST activity, and creatinine and urea
nitrogen concentrations can be elevated in dogs with T. cruzi
infection, especially those that are at risk of death from severe
acute myocarditis. Rarely, moderate to severe hypoalbuminemia
with or without hyperglobulinemia occurs in acutely affected
dogs.16,31 Serum troponin I levels rise slowly in infected dogs
and peak at around 10 to 30 mg/mL by 21 DPI. Serum troponin
I levels are elevated in dogs infected after 6 months of age but
usually not to such high levels.
Urinalysis
There are no specific findings reported on urinalysis in dogs
with Chagas’ disease.
Electrocardiography
The ECG of dogs with severe Chagas’ myocarditis may show sinus
tachycardia, decreased R-wave amplitude, axis shifts, T-wave

B
FIGURE 78-4  Electrocardiographic findings in dogs with Chagas’ disease. A, Second-degree heart block and depressed QRS complexes. (From Barr SC. Canine Chagas’ disease (Ameri-
can trypanosomiasis) in North America. Vet Clin North Am. 2009;39:1055-1064.)B, Multiform ventricular premature complexes in an 11-year-old, neutered male Labrador retriever present-
ing for collapse. Recording speed 25 mm/s, sensitivity 10 mm/mV.
inversion, ventricular arrhythmias, and conduction abnormali-
ties, including first-, second-, and third-degree atrioventricular
block and right bundle branch block (Figure 78-4).
Diagnostic Imaging
Plain Radiography
Plain thoracic radiographs in dogs with chronic Chagas’ myo-
carditis may reveal cardiomegaly or evidence of congestive heart
failure (pulmonary edema or pleural effusion) (Figure 78-5).
Sonographic Findings
Echocardiograms of puppies with acute myocarditis are usu-
ally within normal limits. Echocardiographic abnormalities in
dogs with chronic Chagas’ myocarditis include right ventricular
dilation with progression to include a loss of left ventricular
function with decreased fractional shortening, reduced ejec-
tion fraction, reduced left ventricular free wall thickness, and
increased end-systolic volume (Figure 78-6).
Microbiologic Tests
Diagnostic assays for trypanosomiasis in dogs and cats are
described in Table 78-1.
Cytologic Examination
During acute disease, trypomastigotes may be detected on blood
smear examination (see Figure 78-2). However, only a few par-
asites may be present on the entire slide, demanding diligent
examination, or some form of concentration technique may be
used. High-power (400×) examination of the buffy-coat layer
from a centrifuged microhematocrit tube may reveal the motile
parasites. Examination of a thick-film buffy-coat smear stained
with either Wright or Giemsa stains is more sensitive than
examination of a blood smear. A highly effective concentra-
tion technique involves pelleting trypomastigotes from plasma
(obtained by centrifugation of 10 to 50 mL of heparinized blood
at 800 × g for 10 minutes) by further centrifugation (8000 × g
for 15 minutes). The pellet from the final centrifugation may
be examined microscopically after staining or be submitted for
PCR analysis or culture. The larger the volume of the blood
specimen collected, the greater the likelihood that organisms
will be detected. Trypomastigotes may also be found on cyto-
logic examination of lymph node aspirates and in abdominal
CHAPTER 78  Trypanosomiasis 763
effusions. Amastigotes were described in the lymph node of
one affected dog that were cytologically indistinguishable from
those of Leishmania.31
Culture
Trypanosoma cruzi can be cultured from blood in liver infu-
sion tryptose (LIT) growth medium. However, several weeks are
required before epimastigotes grow in this medium, which is too
long to be helpful for making treatment decisions. Although cul-
ture is more sensitive than blood smear examination, false-neg-
ative results can still occur; in humans the sensitivity of blood
culture is estimated to be only 50%.22 Culture of trypanosomes
is a specialized procedure that is not routinely available in vet-
erinary diagnostic laboratories.
Serologic Diagnosis
Serology is extremely useful for the diagnosis of Chagas’ dis-
ease, especially during the indeterminate and chronic phases
when trypomastigotes are very difficult to detect.34 Indirect fluo-
rescent antibody, ELISA, and radioimmunoprecipitation assays
are most commonly used. A rapid immunochromatographic
dipstick test has also been developed.35 Serologic tests confirm
the presence of antibodies to T. cruzi, but most cross-react with
antibodies to Leishmania. Further, in rare situations in dogs,
the clinical signs of Chagas’ disease and leishmaniasis overlap
sufficiently that it is necessary to go to considerable lengths to
establish a diagnosis.31 Therefore a detailed history of the likeli-
hood of exposure to Leishmania must be known in order to
accurately interpret serologic results. A higher antibody titer to
Trypanosoma than to Leishmania may support a diagnosis of
trypanosomiasis. Positive serology in association with consistent
clinical signs is the most common means of diagnosis of Chagas’
disease in dogs. If possible, two different serologic assays should
be used to confirm a positive titer, especially in regions of low
prevalence. The serum titer usually becomes positive by 21 DPI,
when parasitemia is declining, and persists for the life of the
animal irrespective of whether clinical signs develop.30
Molecular Diagnosis Using the Polymerase Chain Reaction
PCR assays, which detect DNA of T. cruzi in various clinical
specimens (blood, plasma pellets, lymph node aspirates, or
ascites fluid), can be highly specific for T. cruzi but can have
764 SECTION 4  Protozoal Diseases
A
B
FIGURE 78-5  Right lateral (A) and dorsoventral (B) thoracic radiographic images of
a dog with trypanosomiasis and mild generalized cardiomegaly. The pulmonary veins are
slightly larger than the corresponding artery, and a slight interstitial pattern was present
throughout the lungs.
variable and sometimes low sensitivity for diagnosis of chronic
infections unless multiple specimens are tested.34 Real-time
PCR assays have been developed for diagnosis of human Cha-
gas’ disease that detect repetitive sequences found in very high
copy number within T. cruzi.36 Because each parasite may
have more than 100,000 copies of the sequence, the sensitivity
of the assay is increased.22 The sensitivity of PCR assays var-
ies depending on the T. cruzi strain involved.36,37 PCR may
be useful to confirm infection when organisms are not seen
on blood smears. Currently, PCR assays for T. cruzi are not
widely available from veterinary diagnostic laboratories on a
commercial basis, but have been used for diagnosis of canine
Chagas’ disease.31
A
C
FIGURE 78-6  Two dimensional (A) and M-mode (B) transthoracic echocardiogram
images obtained in a right parasternal short-axis view and long-axis view (C) from a dog
with Chagas’ disease documenting significant left ventricular enlargement and mild right
ventricular enlargement.
Pathologic Findings
Gross Pathologic Findings
Gross pathologic findings in dogs with Chagas’ disease include
lymphadenopathy, evidence of congestive heart failure (ascites,
pleural effusion, or congestion of viscera), and biventricular car-
diac enlargement with thinning of the ventricular wall. Often the
right side of the heart is more enlarged than the left. Hemorrhages
and pale foci may be identified in the myocardium.
 CHAPTER 78  Trypanosomiasis 765

TABLE 78-1
Diagnostic Assays Currently Available for Trypanosomiasis in Dogs and Cats
Assay
Cytologic examination
Trypanosoma culture
Antibody serology
(immunofluorescent
antibody or ELISA)
Real-time PCR assays
Specimen Type
Aspirates of lymph nodes,
spleen, bone marrow,
blood smears, or buffy-
coat preparations
Blood or buffy-coat
preparations, bone
marrow
Serum
Whole blood, buffy coats,
splenic or lymph node
aspirates, CSF
Target Performance
Trypanosoma Organisms may be few in number and in chronic
trypomastigotes or, trypanosomiasis are often not found.
less commonly,
amastigotes
Trypanosoma spp. Requires special techniques and expertise that are
not widely available. Sensitivity may be low in
chronic infections.
Antibodies to Most infected dogs test positive, but positive test
Trypanosoma spp. results occur in healthy animals, so results must
be interpreted in light of clinical signs. False posi-
tives can occur in dogs exposed to Leishmania or
those vaccinated with Leishmania vaccines.
Trypanosoma DNA May be useful in dogs with suggestive clinical signs
in which organisms cannot be found cytologi-
cally. Sensitivity and specificity depend on assay
design. Quantitative PCR may be useful for evalu-
ation of treatment efficacy. Limited availability.

TABLE 78-2
Antimicrobials Used to Treat Acute Chagas’ Disease in Dogs
Dose Interval Duration
Drug* (mg/kg) Route (hours) (months)
Benznidazole 5-10 PO 24 2
Nifurtimox 2-7 PO 6 3-5
Prednisone 0.5 PO 12 1

*Both benznidazole and nifurtimox are available from the Centers for
Disease Control and Prevention, Atlanta, GA.

Treatment and Prognosis

FIGURE 78-7  Pseudocyst of Trypanosoma cruzi within a myocardiocyte of an infected
dog. H&E stain, 1000× magnification. (From Barr SC. Canine Chagas’ disease (American
trypanosomiasis) in North America. Vet Clin North Am. 2009;39:1055-1064.)
Histopathologic Findings
Histopathologic findings in Chagas’ disease include a severe
diffuse granulomatous myocarditis, large numbers of parasitic
pseudocysts (intracellular collections of amastigotes), and mini-
mal fibrosis (Figure 78-7). Immunohistochemistry does not dis-
tinguish Leishmania from Trypanosoma amastigotes, because of
serologic cross-reactivity.31 Histopathology of the myocardium
in chronic Chagas’ myocarditis is characterized by multifocal
lymphocytic infiltrates, perivasculitis, and marked fibrosis, and
rare, if any, parasitic pseudocysts.4,27
Antimicrobial Treatment
Treatment of dogs with naturally occurring acute Chagas’
myocarditis is poorly documented, since this phase is seldom
recognized. The use of nifurtimox (usually in association with
glucocorticoids)38 or benznidazole39 has been reported in the
dog (Table 78-2). However, the severe adverse effects of nifur-
timox preclude its use, so benznidazole is the drug of choice
(see Chapter 10). Benznidazole is available from the Centers
for Disease Control and Prevention, Atlanta, GA. After treat-
ment with benznidazole, serum antibody titers usually remain
elevated, although they are reported to decline in people.
Treatment of experimentally infected dogs with acute Chagas’
disease with benznidazole reduced or prevented subsequent
fibrosis and arrhythmias, even if parasites were not completely
eliminated.40 Benznidazole-resistant strains of T. cruzi have
been described. Ketoconazole, gossypol, allopurinol, and vera-
pamil have shown promise in other species but are ineffective
for the treatment of Chagas’ disease in dogs. Ravuconazole and
posaconazole also reduce parasite load,41,42 but to date azole
766 SECTION 4  Protozoal Diseases
drugs have not been adopted for treatment of humans because
they lack significant benefit over benznidazole or nifurtimox.22
However, the combination of the antiarrhythmic amiodarone
and an azole (itraconazole or posaconazole) has synergistic
antiprotozoal activity against T. cruzi and shows promise as an
effective treatment for chronic Chagas’ disease in humans.43,44
Most dogs are diagnosed with Chagas’ disease during the
chronic stage. Unfortunately, treatments directed against the
parasite at this stage do not significantly alter the outcome of
disease.45 Treatment should be directed toward the myocar-
dial failure and ventricular arrhythmias, although the latter are
often refractory to drug therapy. Pacemaker implantation may
be required for some dogs with bradyarrhythmias such as third-
degree atrioventricular block. In humans with indeterminate and
chronic symptomatic Chagas’ disease, the benefit of treatment
remains controversial and is still under investigation by means
of a large, prospective clinical trial (the Benznidazole Evaluation
for Interrupting Trypanosomiasis [BENEFIT] trial).36
Prognosis
The prognosis for dogs that develop Chagas’ disease is poor
because of the lack of effective treatments. Dogs diagnosed at
an older age (mean of 9 years) survive longer (30 to 60 months)
than dogs diagnosed at a younger age (mean of 4.5 years), which
survive only up to 5 months postdiagnosis.11
Immunity and Vaccination
Recovery from T. cruzi infection is not associated with protective
immunity. No vaccine is available.
Prevention
The risk of infection by T. cruzi can be reduced by limiting con-
tact between dogs and vectors and possible reservoir hosts (rac-
coons, opossums, armadillos, and skunks). Dogs should not be
fed meat from reservoir hosts. Kennels and surrounding struc-
tures (chicken houses, wood piles) in endemic areas should be
sprayed monthly with a residual insecticide. Dog housing should
be upgraded to remove vector nesting sites. Applications of
fipronil on the coats of dogs do not appear to prevent infections
in dogs or reduce the feeding of vectors,46 but deltamethrin-
treated collars do reduce feeding by Triatoma infestans.47,48
Blood donor dogs should be serologically screened to determine
previous exposure to T. cruzi. In highly endemic regions (south-
ern Texas), bitches should be screened serologically, and posi-
tive animals should not be bred.
Public Health Aspects
American trypanosomiasis is a major human health problem in
South and Central America and is increasingly recognized in
Mexico.8,49 It is primarily a problem in impoverished regions
where humans live in rural areas. Dogs are considered a reser-
voir for human infection in these regions because they maintain
high levels of parasitemia and are a preferred host for triatomine
insects. Cats may also act as a reservoir for human infection
in South America.50 Fewer than one third of infected humans
ultimately develop Chagas’ disease.22
To date, fewer than 10 human cases of Chagas’ disease
involving transmission by vectors have been reported in the
United States (4 in Texas, and 1 each in California, Tennessee,
and Louisiana). By far the largest number of people infected
with T. cruzi in the United States (estimated at approximately
100,000 people) have emigrated from endemic regions such as
Mexico and Central America. Consequently, reports of cases
associated with transmission by blood transfusion continue to
rise.51,52 Imported cases in tourists who return to the United
States have not been described. There are probably several rea-
sons why only a handful of naturally acquired cases have been
reported in the United States. First, North American vector spe-
cies are poorly adapted to living in houses and do not defecate
on the host after a blood meal. Higher standards of housing in
North America than in parts of Central and South America may
also prevent vectors from nesting in human dwellings. Third,
some human cases of Chagas’ disease in North America may be
overlooked because of a low level of suspicion.
Although the risk of a human acquiring infection from an
infected dog is extremely low, the severity and difficulty of
treating disease in humans makes this disease of considerable
public health significance. Veterinarians should be careful when
handling blood samples from infected dogs and warn labora-
tory staff of the potential infectivity of the samples. Accidental
needle-stick injuries when administering therapy to or collecting
specimens from infected dogs should be reported immediately
to the CDC.
Overview of African Trypanosomiasis in Dogs
First Described: Reports of African trypanosomiasis date back
to Egyptian times. Trypanosomes were identified as the
cause in 1895, when they were detected in cattle by a
Scottish microbiologist, David Bruce (after whom Brucella
was named).53
Cause: Trypanosoma brucei rhodesiense, Trypanosoma con-
golense, Trypanosoma evansi (protozoan parasites, family
Trypanosomatidae)
Affected Hosts: Dogs and a variety of other mammalian host
species. Humans are infected with T. brucei gambiense and
T. brucei rhodesiense.
Geographic Distribution: Central Africa (T. brucei, T. congolense);
T. evansi is found in Africa as well as in Asia, Latin America,
and the Canary Islands in Spain.
Mode of Transmission: A bite from the tsetse fly (T. brucei or
T. congolense) or mechanical transmission by hematopha-
gous flies (T. evansi)
Major Clinical Signs: Lethargy, inappetence, weight loss, pal-
lor, peripheral edema, ascites, hematemesis, hemorrhagic
diarrhea, uveitis, corneal edema, neurologic signs due to
meningoencephalitis
Differential Diagnoses: These include canine monocytic
ehrlichiosis, babesiosis, leishmaniosis, and systemic
immune-mediated diseases. Canine distemper virus
infection and neosporosis are important differential diag-
noses in dogs with meningoencephalitis.
Human Health Significance: Although most Trypanosoma
species that cause African trypanosomiasis in dogs do
not infect humans, T. brucei rhodesiense does, so caution is
advised when handling tissues or clinical specimens from
dogs or cats with suspected trypanosomiasis.

AFRICAN TRYPANOSOMIASIS
African trypanosomiasis is caused by Trypanosoma brucei and
Trypanosoma congolense. These organisms are transmitted by
tsetse flies (Glossina species), which are only found in Africa. Try-
panosoma brucei includes Trypanosoma brucei brucei, T. brucei
gambiense, and T. brucei rhodesiense, which are indistinguish-
able morphologically and are referred to as “T. brucei complex”
organisms. T. evansi also belongs to this group (see later). The
epidemiology, host range, and clinical features of disease caused
by these T. brucei complex organisms differ. Trypanosoma brucei
gambiense and T. brucei rhodesiense cause “sleeping sickness” in
humans from Africa (West African and East African trypanoso-
miasis, respectively) (Figure 78-8). A variety of animal reservoirs
of T. brucei rhodesiense have been identified, but an animal res-
ervoir for T. brucei gambiense has not.54 Along these lines, only
naturally occurring infections with T. brucei rhodesiense , and
not T. brucei gambiense, have been reported in dogs.55 Trypano-
soma brucei brucei and T. congolense cause trypanosomiasis in a
variety of domestic animal species (“nagana”) but do not infect
humans. Dogs are especially susceptible to T. congolense infec-
tion. Four types of T. congolense have been identified—savan-
nah, forest, Tsavo, and Kilifi—which may vary in pathogenicity.
There are several reports of travel-related infections with T. con-
golense in dogs,56-58 sometimes in association with a long period
of latency.57 It has been suggested that African dogs may exhibit a
degree of “trypanotolerance,” whereas dogs imported into Africa
may be more susceptible to infection and disease.59
African trypanosomes exist only as a trypomastigote in the
blood of the mammalian host; in contrast to Chagas’ disease,
there is no amastigote phase. Epimastigotes develop in the salivary
FIGURE 78-8  Map showing geographic distribution of human African trypanosomiasis (green countries) and the number of cases reported in natives in the year 2009. Trypanosoma brucei
gambiense is found in western Africa and Trypanosoma brucei rhodesiense is found in eastern Africa. The line divides the geographic distribution of the two forms of disease. T. brucei brucei,
which infects dogs, occurs throughout both regions. The hashed lines indicate the countries in which tsetse flies are found. (Modified from Brun R and Blum J. Human African Trypanosomiasis.
Inf Dis Clin North Am. 2012;26:261-273.)
CHAPTER 78  Trypanosomiasis 767
glands of the tsetse fly, and the organism is transmitted in saliva
when it feeds on the mammalian host. Transmission to the mam-
malian host results in hemolymphatic spread of the organism over
weeks to months with signs that relate to proliferation of mono-
nuclear phagocytes and increased vascular permeability. T. brucei
complex organisms are well known for their ability to evade the
host immune response through extensive variation of their outer
surface glycoproteins (known as variant antigen types, or VATs).
The hemolymphatic phase may be followed by the development
of meningoencephalitis in some animals. In humans, meningoen-
cephalitis is most apparent with T. brucei gambiense infections
and results in tremors and progressive daytime somnolescence
(hence the term “sleeping sickness”). Pancarditis with arrhyth-
mias and congestive heart failure is a more typical outcome of T.
brucei rhodesiense infection in humans.
The clinical signs of African trypanosomiasis in dogs include
persistent fever, lethargy, anorexia, weight loss, pallor, muco-
purulent oculonasal discharge, lymphadenopathy, hepatospleno-
megaly, variable peripheral edema, abdominal distention due to
ascites, petechial hemorrhages, signs of pancarditis, and ocular
signs such as unilateral or bilateral uveitis, corneal edema, and/
or keratitis (which can mimic “blue eye” due to canine adeno-
virus-1). Hemorrhagic vomiting and diarrhea can occur in dogs
infected with T. congolense.58,60 Neurologic signs (seizures, trem-
ors, opisthotonos, and hyperreflexia) due to meningoencephalitis
have also been described in dogs infected with T. congolense.58
Laboratory abnormalities in dogs with African trypanosomi-
asis have been most well described for T. congolense infections.
The most common findings are regenerative or nonregenerative
anemia, leukocytosis or leukopenia, and thrombocytopenia.57,58,60
Serum biochemistry findings include increased serum liver
768 SECTION 4  Protozoal Diseases
enzyme activities, azotemia, hyperglobulinemia, and hypoalbu-
minemia,57,58 but one dog had no significant abnormal serum
biochemistry findings.56
The primary means of diagnosis is cytologic detection of the
organism in aspirates, body fluids, or blood smears. Identification
of the infecting species requires PCR and sequencing. The treat-
ment of choice for dogs is diminazene aceturate, but this may not
always be effective or available. Treatment of the hemolymphatic
disease in humans is with pentamidine isethionate (gambiense) or
suramin (rhodesiense), whereas disease of the central nervous sys-
tem is treated with the arsenical melarsoprol, but these drugs can
have significant adverse effects. Pentamidine was used with appar-
ent success to treat one dog with African trypanosomiasis.61 Pre-
vention is through the use of insect repellants and indoor housing.
OTHER TRYPANOSOMA INFECTIONS
Other Trypanosoma species that can infect dogs (and to a lesser
extent, cats) include Trypanosoma evansi and Trypanosoma
caninum.
T. evansi causes surra (= “rotten”) and is endemic in large
parts of Asia, Africa, Latin America, and the Canary Islands
of Spain. It causes disease in a variety of mammalian host spe-
cies that include horses, cattle, dogs, and cats, but not humans.
T. evansi is very closely related to T. brucei and may have
derived from T. brucei as a result of partial or complete loss
CASE EXAMPLE
Signalment: “Duke,” a 5-year-old male beagle mix dog from
San Antonio, TX
History: Duke’s owner reported a 7-day history of inappetence,
lethargy, and decreased exercise tolerance. Two months
earlier, the dog had been diagnosed with dirofilariasis and
was treated with melarsomine dihydrochloride using a split
protocol. He had not traveled out of his local area. He was
up to date on vaccinations, which included vaccines for
distemper, hepatitis, parvovirus, and rabies.
Current medications: Monthly oral ivermectin/pyrantel
heartworm preventative.
Physical Examination:
Body weight: 19.9 kg.
General: Lethargic. T = 101.7°F (38.7°C), HR = 142 beats/min,
RR = 48 breaths/min, mucous membranes pink, CRT = 1 s.
Integument, Eyes, Ears, Nose, and Throat: Full, shiny
haircoat. No evidence of ectoparasites was detected. No
other significant abnormalities were noted.
Musculoskeletal: Body condition score was 6/9.
Cardiovascular: An irregular rhythm and a grade 3/6 systolic
left apical murmur were detected on thoracic auscultation.
Strong femoral pulses were palpated bilaterally.
Respiratory: Mild tachypnea was identified with normal breath
sounds and normal respiratory effort.
Gastrointestinal, Genitourinary, and Lymph Nodes: No
abnormalities were detected.
Laboratory Findings:
CBC:
PCV 48.3% (31%-56%)
MCV 66.4 fL (60-77 fL)
of kinetoplast DNA, which stops it from developing within the
insect vector. Instead, the organism is transmitted mechanically
by biting flies. Ingestion of infected meat may also lead to infec-
tion. Because populations of biting flies are greatest during the
rainy season, a seasonal incidence of disease has been observed
in dogs.62 Travel-related disease has been described.63,64 The
disease has an acute course; clinical signs and laboratory abnor-
malities resemble those of African trypanosomiasis, with leth-
argy, fever, anorexia, weight loss, pallor, lymphadenopathy,
vomiting, conjunctivitis, uveitis, corneal edema, and peripheral
edema, as well as leukopenia, thrombocytopenia, and anemia on
the CBC.62-65 Hyperglobulinemia and proteinuria have also been
reported.64 Some dogs develop neurologic signs late in the course
of disease due to nonsuppurative meningoencephalitis.64 Defini-
tive diagnosis of infection is based on cytologic examination
of blood smears and/or PCR assay. Treatment is usually with
diminazene aceturate or suramin, although diminazene aceturate
is considered less effective against T. evansi than T. congolense
infection.64 Untreated infection is usually fatal in dogs.
Trypanosoma caninum is a recently discovered Trypanosoma
species that has only been identified in dogs from South America,
many of which have been apparently healthy.66,67 The pathogenic-
ity of this organism requires further study. Importantly, infection
with T. caninum leads to false-positive serologic test results for
Leishmania, which can confound control methods that rely on cull-
ing of dogs that are seropositive for Leishmania (see Chapter 74).
MCHC 35 g/dL (32-36 g/dL)
WBC 13,100 cells/µL (6000-17,000 cells/µL)
Neutrophils 8777 cells/µL (3000-11,500 cells/µL)
Lymphocytes 2096 cells/µL (1000-4800 cells/µL)
Monocytes 1179 cells/µL (150-1250 cells/µL)
Platelets 238,000 platelets/µL (200,000-500,000 platelets/µL)
Serum Chemistry Profile:
Sodium 144 mmol/L (139-147 mmol/L)
Potassium 3.9 mmol/L (3.3-4.6 mmol/L)
Chloride 118 mmol/L (107-118 mmol/L)
Bicarbonate 22 mmol/L (20-28 mmol/L)
Phosphorus 3.6 mg/dL (2.9-6.2 mg/dL)
Calcium 9.8 mg/dL (9.3-11.8 mg/dL)
BUN 19 mg/dL (5-29 mg/dL)
Creatinine 1.27 mg/dL (0.3-2.0 mg/dL)
Glucose 108 mg/dL (60-135 mg/dL)
Total protein 5.5 g/dL (5.7-7.8 g/dL)
Albumin 2.6 g/dL (2.4-3.6 g/dL)
Globulin 2.9 g/dL (1.7-3.8 g/dL)
ALT 136 U/L (10-130 U/L)
ALP 103 U/L (21-147 U/L)
GGT 11 U/L (0-25 U/L)
Cholesterol 199 mg/dL (120-247 mg/dL)
Total bilirubin 0.2 mg/dL (0-0.8 mg/dL)
Magnesium 2.1 mg/dL (1.7-2.1 mg/dL)
Urinalysis: SGr 1.012; pH 7.5, trace protein (SSA), no bilirubin,
no glucose, no WBC/HPF, 0-1 RBC/HPF.
Heartworm Antigen Test: Negative.
Cardiac Troponin I: 0.69 ng/mL (<0.2 ng/mL).
Chagas Immunofluorescent Antibody Titer: Positive at
1:160.

Imaging Findings:
Thoracic Radiographs: The cardiac silhouette was mildly,
generally enlarged. The pulmonary veins were mildly larger
than the corresponding artery. A mild pulmonary interstitial
pattern was present.
Echocardiogram: The left ventricular internal dimensions
were enlarged, and systolic function was reduced (fractional
shortening 16.1%, area shortening 26.9%; normal is >25%
and >50%, respectively). Moderate mitral regurgitation was
documented, and the left atrium was enlarged. The right
ventricle was mildly dilated. Pulmonic and aortic velocities
were within normal limits. No heartworms were visualized.
Additional Testing: Electrocardiogram: Sinus rhythm with
frequent, multiform ventricular premature complexes.
Blood pressure: Systolic blood pressure was 115 mm Hg.
Diagnosis: American trypanosomiasis (Chagas’ disease).
Treatment: Medication to address cardiac enlargement,
dysfunction, and arrhythmias included pimobendan (0.25
mg/kg PO q12h), enalapril (0.5 mg/kg PO q12h), and sotalol
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33. Quijano-Hernandez IA, Castro-Barcena A, Aparicio-Burgos E,
et  al. Evaluation of clinical and immunopathological features of
different infective doses of Trypanosoma cruzi in dogs during the
acute phase. Scientific World Journal. 2012;2012:635169.
34. Araujo FM, Bahia MT, Magalhaes NM, et al. Follow-up of experi-
mental chronic Chagas’ disease in dogs: use of polymerase chain
reaction (PCR) compared with parasitological and serological
methods. Acta Trop. 2002;81:21-31.
35. Rosypal AC, Hill R, Lewis S, et  al. Evaluation of a rapid immu-
nochromatographic dipstick test for detection of antibodies to
Trypanosoma cruzi in dogs experimentally infected with isolates
obtained from opossums (Didelphis virginiana), armadillos (Dasy-
pus novemcinctus), and dogs (Canis familiaris) from the United
States. J Parasitol. 2011;97:140-143.
36. Moreira OC, Ramirez JD, Velazquez E, et al. Towards the estab-
lishment of a consensus real-time qPCR to monitor Trypano-
soma cruzi parasitemia in patients with chronic Chagas disease
cardiomyopathy: a substudy from the BENEFIT trial. Acta Trop.
2013;125:23-31.
37. Veloso VM, Guedes PM, Andrade IM, et al. Trypanosoma cruzi:
blood parasitism kinetics and their correlation with heart parasit-
ism intensity during long-term infection of Beagle dogs. Mem Inst
Oswaldo Cruz. 2008;103:528-534.
38. Andrade ZA, Andrade SG, Sadigursky M, et al. Experimental Cha-
gas’ disease in dogs. A pathologic and ECG study of the chronic inde-
terminate phase of the infection. Arch Pathol Lab Med. 1981;105:
460-464.
39. Viotti R, Vigliano C, Armenti H, et al. Treatment of chronic Cha-
gas’ disease with benznidazole: clinical and serologic evolution of
patients with long-term follow-up. Am Heart J. 1994;127:151-162.
40. Caldas IS, da Matta Guedes PM, Dos Santos FM, et al. Myocardial
scars correlate with electrocardiographic changes in chronic Try-
panosoma cruzi infection for dogs treated with benznidazole. Trop
Med Int Health. 2013;18:75-84.
41. Diniz Lde F, Caldas IS, Guedes PM, et al. Effects of ravuconazole
treatment on parasite load and immune response in dogs experi-
mentally infected with Trypanosoma cruzi. Antimicrob Agents
Chemother. 2010;54:2979-2986.
42. Veiga-Santos P, Barrias ES, Santos JF, et al. Effects of amiodarone
and posaconazole on the growth and ultrastructure of Trypano-
soma cruzi. Int J Antimicrob Agents. 2012;40:61-71.
43. Paniz-Mondolfi AE, Perez-Alvarez AM, Lanza G, et al. Amiodarone
and itraconazole: a rational therapeutic approach for the treatment
of chronic Chagas’ disease. Chemotherapy. 2009;55:228-233.
44. Benaim G, Sanders JM, Garcia-Marchan Y, et al. Amiodarone has
intrinsic anti–Trypanosoma cruzi activity and acts synergistically
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45. Santos FM, Lima WG, Gravel AS, et al. Cardiomyopathy progno-
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46. Gurtler RE, Ceballos LA, Stariolo R, et al. Effects of topical appli-
cation of fipronil spot-on on dogs against the Chagas disease vector
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47. Reithinger R, Ceballos L, Stariolo R, et al. Chagas disease control:
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48. Reithinger R, Ceballos L, Stariolo R, et  al. Extinction of experi-
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49. Estrada-Franco JG, Bhatia V, Diaz-Albiter H, et  al. Human Try-
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50. Cardinal MV, Lauricella MA, Ceballos LA, et al. Molecular epide-
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infection in a dog in France. Vet Rec. 2011;168:590.
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60. Ezeokonkwo RC, Ezeh IO, Onunkwo JI, et al. Comparative hae-
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63. Hellebrekers LJ, Slappendel RJ. Trypanosomiasis in a dog imported
in The Netherlands. Vet Q. 1982;4:182-186.
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and immunological aspects of experimental infection with Trypano-
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66. Madeira MF, Sousa MA, Barros JH, et al. Trypanosoma caninum
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Parasitology. 2010;137:1653-1660.
CHAPTER 79
Giardiasis
Michael R. Lappin
Overview of Giardiasis
First Described: Trophozoites that were likely Giardia spp.
were first described in 1681 by Antonie van Leeuwenhoek
(who found the parasite in his own stools1) and the genus
was established in the early 1900s. Infections of dogs and
cats were first described in the 1960s.2
Cause: Giardia duodenalis (multiple genetic assemblages); a
flagellate protozoan (phylum Sarcomastigophora)
Transmission: Fecal-oral
Affected Host Species: Dogs and cats are the definitive hosts
for several different Giardia assemblages and pass either
trophozoites (diarrhea) or cysts (diarrhea or normal stool)
in feces.
Intermediate Hosts: Although there are no intermediate
hosts, there are multiple potential transport hosts that
may carry Giardia spp. cysts and indirectly infect dogs
or cats.
Geographic Distribution: Worldwide
Major Clinical Signs: Most dogs and cats harbor subclinical
infections. Acute watery diarrhea that can contain mucus
can occur. Steatorrhea can be observed. Chronic or inter-
mittent diarrhea and weight loss may be noted in animals
with concurrent infections or immunocompromise.
Differential Diagnoses: All causes of small bowel diarrhea
including Isospora spp., Cryptosporidium spp., bacterial
infections such as salmonellosis, nematode infections,
dietary intolerance, exocrine pancreatic insufficiency, and
inflammatory bowel disease
Human Health Significance: Giardia assemblages that infect
dogs (assemblage C and D) or cats (assemblage F) are usu-
ally not associated with clinical disease in humans but can
be detected in the feces of immunocompromised people.
Human assemblages A and B can be found in feces of dogs
and cats when animals share human environmental fea-
tures (such as contaminated water). However, it is unlikely
that new human infections with Giardia assemblage A or
B infections are acquired from the feces of dogs or cats.
Etiologic Agent and Epidemiology
Giardia spp. are protozoans in the flagellate group that have
been recognized in the feces of animals and man for many
years.3 Giardia spp. occurs in two forms, the trophozoite and
the cyst (Figure 79-1). The trophozoite is the active, motile
form; it is approximately 15 µm long and 8 µm wide and has
a teardrop shape and a ventral adhesive disk (Figure 79-2).
Trophozoites are almost never found in normal feces and are
susceptible to many environmental conditions; thus, they usu-
ally are not associated with transmission among animals. The
12-µm long and 7-µm wide cyst is the environmentally resis-
tant stage primarily responsible for transmission. The cyst con-
tains two incompletely separated but formed trophozoites, the
axoneme, fragments of the ventral disks, and as many as four
nuclei (Figure 79-3). The cyst can survive several months out-
side the host in wet and cool conditions but is susceptible to
desiccation in dry and hot conditions.
Giardia spp. are transmitted by the direct ingestion of fecal
cysts or by indirect ingestion of contaminated water, food,
transport hosts, infected prey species, or fomites. The prepatent
period (time between ingestion and appearance of cysts in the
feces) in cats ranges from 5 to 16 days (mean around 10 days)
and in dogs ranges from 4 to 12 days (mean around 8 days).
Shedding of Giardia cysts by cats may fluctuate from undetect-
able to concentrations of more than 1 million cysts/g of feces
over periods as short as 2 to 7 days.4 Young dogs shed an aver-
age of 2000 cysts per gram of feces; the mean cyst count per
gram of feces for all infected dogs (regardless of age) was 706.5
Most infections are self-limited with cessation of cyst shedding
in 27 to 35 days in dogs and cats. However, some infected ani-
mals shed cysts for several months.
Although there are many Giardia spp., G. duodenalis
(syn. G. intestinalis or G. lamblia) is the species that infects
people, dogs, and cats.6 Based on genetic analyses, there are at
least seven distinct assemblages (A-G) within G. duodenalis.
Assemblage A has been amplified from feces of infected
humans, dogs, and cats as well as many other mammals.
Assemblage B is most commonly amplified from human feces
but is occasionally amplified from dog feces. Overall, most
dogs are infected with the species-specific assemblages C and
D, and cats are infected with assemblage F.6 It has been pro-
posed that these assemblages be recategorized as separate
species of Giardia (Table 79-1), but this has not yet been
unanimously accepted.7
Giardia infections in cats and dogs have been reported in
many prevalence studies, case reports, and treatment studies
from around the world. Infection rates vary with the assay used
and whether or not diarrhea is occurring. However, in general
they are approximately 5% to 20% in dogs and cats.3 In most
studies there is no association between the presence of Giardia
in the feces and clinical signs, because of the high prevalence
of subclinical infections.3,8 Younger or immunosuppressed ani-
mals and those living in crowded environments are at the highest
risk of showing clinical signs of disease.
771
772 SECTION 4  Protozoal Diseases

FIGURE 79-3  Giardia spp. cysts after concentration by sugar centrifugation (400×
magnification). The cysts are approximately 12 µm long and 7 µm wide. (Courtesy of 
Dr. Lora Ballweber, Colorado State University. In from Tangtrongsup S, Scorza V. Update
on the diagnosis and management of Giardia spp. infections in dogs and cats. Topics in
Companion Animal Medicine. 2010;25:155-162.)

TABLE 79-1
Proposed and Existing Nomenclature in the Genus Giardia
Species Hosts
Giardia duodenalis (assemblage A*) Wide range of domestic
FIGURE 79-1  Life cycle of Giardia duodenalis.
and wild mammals
including humans
Giardia enterica (assemblage B) Humans and other pri-
mates, dogs, some spe-
cies of wild mammals
Giardia canis (assemblage C/D) Dogs and other canids
Giardia cati (assemblage F) Cats
Giardia bovis (assemblage E) Cattle, other hoofed
livestock
Giardia simondi (assemblage G) Rats
Giardia muris Rodents
Giardia microti Muskrats and voles
Giardia ardeae Birds
Giardia psittaci Birds
Giardia agilis Amphibians

Modified from Thompson RCA, Smith A. Zoonotic enteric protozoa.

Vet Parasitol. 2011;182(1):70-78.
*The Giardia assemblages listed currently fall within the single species
Giardia duodenalis

FIGURE 79-2  Electron micrograph of a Giardia trophozoite. (Courtesy of Bayer
Animal Health. In Tangtrongsup S, Scorza V. Update on the diagnosis and manage-
ment of Giardia spp. infections in dogs and cats. Topics in Companion Animal Medicine.
2010;25:155-162.)
Clinical Features
Signs and Their Pathogenesis
After Giardia spp. cysts are ingested by a susceptible host, gas-
tric acid and pancreatic enzyme exposure in the duodenum
induces release of the two trophozoites, which mature quickly
and attach to the brush border of the villous epithelium by
means of their ventral adhesive disk and a number of parasitic

surface molecules, including the giardins (alpha, beta, delta, and
gamma giardins) and a network of contractile proteins. In dogs,
trophozoites are found from the duodenum to the ileum; in cats,
they predominate in the jejunum and ileum. Trophozoites multiply
by binary fission and then encyst by an unknown mechanism.
Contamination of fomites, which can include arthropods and
the coats of animals, or food and especially water sources by
Giardia cysts is an important mode of transmission, because
cysts remain viable for days to weeks in the environment when
conditions are cool and moist.
Although the pathogenesis of Giardia is not completely
understood, in vitro and in vivo studies suggest it is multifacto-
rial with a combination of intestinal malabsorption and hyperse-
cretion as the primary mechanisms associated with development
of diarrhea. The host-parasite interaction leads to the upregula-
tion of genes involved in the apoptotic cascade and in the for-
mation of reactive oxygen species.9 The induction of apoptosis
increases gastrointestinal permeability, allowing luminal anti-
gens to activate host immune-dependent pathologic pathways.
Giardia also alters epithelial claudin proteins that are compo-
nents of tight junctions.9 However, Giardia can cause clinical
signs in the absence of villous atrophy or other signs of mucosal
injury.9 Giardia infection stimulates chloride secretion and results
in a diffuse loss of brush border microvillus length, which causes
epithelial maldigestion and malabsorption of glucose, sodium,
and water and reduced disaccharidase activity. Mild to moder-
ate infiltrates of intraepithelial lymphocytes can be recognized
and may be associated with the sodium and glucose malabsorp-
tion. Loss of epithelial barrier function may have the potential to
lead to chronic intestinal disorders, but the mechanisms remain
unclear.9 Mast cell hyperplasia may also contribute to the loss of
epithelial barrier function. It is unknown why some people and
animals maintain chronic, subclinical infections, but this is likely
related to both host and parasite factors.
Although most cats and dogs that shed Giardia do not show
clinical signs of disease, Giardia infection can induce illness in
some animals. The primary clinical signs of giardiasis include
chronic diarrhea and weight loss. The diarrhea is usually mucoid,
pale, and soft and has a strong odor; steatorrhea may be present as
well.3 The presence of blood in the stool is uncommon unless co-
infections with other agents such as Ancylostoma spp., Isospora
spp., Tritrichomonas foetus, or Clostridium perfringens occur.
Fever or vomiting is almost never recognized in dogs or cats with
giardiasis. Co-infections with other agents such as Cryptosporid-
ium spp. or T. foetus may potentiate diarrhea in dogs or cats.10,11
It is currently unknown whether the different assemblages induce
different clinical signs in cats and dogs or if there are strains within
a given assemblage that are more pathogenic than others.
Diagnosis
Laboratory Abnormalities
Giardia spp. infection alone is rarely associated with CBC or
serum biochemical panel abnormalities.
Diagnostic Imaging
Abdominal radiographic abnormalities are uncommon and non-
specific and, when present, are suggestive of diffuse enteritis.
Microbiologic Testing
Because of the small size of Giardia cysts and the high sub-
clinical infection rate, giardiasis is commonly misdiagnosed,
CHAPTER 79  Giardiasis 773
underdiagnosed, or overdiagnosed.3,12 Yeasts are easily mistaken
for Giardia, which leads to false-positive fecal flotation results.
Giardia cysts are shed intermittently, and repeated fecal analysis
may be needed to detect cysts. In addition, cysts can deteriorate
in fecal flotation solutions, leading to false-negative results.12 A
variety of different Giardia assays have been evaluated for use
in cats and dogs (Table 79-2). There is no one test that can be
performed on a single fecal specimen that has 100% sensitivity.
The following is a discussion of the currently available diagnostic
methods.
Cytologic Examination
Feces can be evaluated for the presence of Giardia trophozoites by
wet mount examination. Approximately 2 mm × 2 mm of fresh
diarrheic feces or mucus is mixed with a drop of normal saline
solution (warmed to body temperature) on a microscope slide and
a coverslip placed. At 100× magnification, trophozoites are rec-
ognized by their “falling leaf” motion. Evaluation for structural
characteristics is then performed at 400× magnification. Because
trophozoites may be associated with mucus, the only visible motil-
ity may be the flagella. Although Giardia trophozoites can be con-
fused with tritrichomonads that are similar in size, they can be
differentiated from tritrichomonads based on their motility and
morphology. In contrast to Giardia spp., tritrichomonads have
an undulating membrane and a rolling form of motility, lack a
concave surface, and have only a single nucleus (see Chapter 80).3
Further testing of feces for Giardia antigen, Giardia DNA by PCR
assay, or fecal culture or PCR assay for Tritrichomonas foetus can
be used to differentiate the organisms if cytology is inconclusive.
Once Giardia is detected in a direct smear or wet mount based on
motility, further cytologic examination with staining should be
performed on a fresh specimen. The application of Lugol’s solu-
tion, methylene blue, or acid methyl green to the wet mount helps
in the visualization of the internal structures of the trophozoites.
Trophozoites are rarely found in solid feces.
Concentration Techniques
Giardia cysts are more likely to be detected after fecal concen-
tration techniques. Sheather’s sugar centrifugation and zinc
sulfate centrifugation are commonly used. In addition to the
identification of Giardia cysts, ova of other common parasites
can easily be identified. Giardia cysts can be confused with yeast
because of similar size, but Giardia cysts have a distinct struc-
ture (see Figure 79-3). Because of the intermittent pattern of
shedding, at least three specimens should be examined over a
period of about 1 week before ruling out Giardia spp. infection
as a cause of diarrhea. If fecal specimens cannot be examined
immediately, storage at 4°C for several days is acceptable, but
specimens should not be frozen.
Duodenal Aspirates
Endoscopic sampling of duodenal secretions and then examina-
tion of the sediment for trophozoites was once considered the
most sensitive technique for detection of Giardia spp. infection
in dogs, but with the advent of immunologic and molecular
techniques, duodenal aspirates are no longer performed. In cats,
because of the location of the parasite in the mid to distal small
bowel, this technique is unlikely to yield positive results.
Direct Immunofluorescence
A monoclonal antibody–based immunofluorescent antibody
(IFA) assay (Merifluor Cryptosporidium/Giardia, Meridian
774 SECTION 4  Protozoal Diseases
TABLE 79-2
Diagnostic Assays Available for Giardiasis in Dogs or Cats
Assay Specimen Type Target
Cytologic examination Feces Giardia trophozoites
and cysts
Centrifugal fecal flotation Feces Giardia cysts
(concentration techniques)
Direct immunofluorescence Feces Giardia cysts
Giardia antigen ELISA assays Feces Giardia cyst antigen
PCR assays Feces Giardia spp. DNA
Diagnostics, Cincinnati, OH) used to detect Giardia spp. cysts
and Cryptosporidium spp. oocysts in human feces has been
applied to feces from dogs and cats in multiple studies.3,13,14
It is believed that Giardia cysts from assemblages C, D, and
F are detected by this assay, but this hypothesis has not been
rigorously assessed using genotyped specimens. The assay gives
both immunologic confirmation (fluorescence) and morpho-
logic evaluation (size and shape) to confirm the presence of
Giardia in feces. In one study, the assay was more sensitive and
specific than zinc sulfate flotation but comparable to an antigen-
detection technique when used on fecal specimens from shelter
dogs.13 Because Cryptosporidium spp. are also associated with
small bowel diarrhea, can exist as co-infections with Giardia,
and are difficult to detect with other techniques, the IFA assay is
used frequently by some clinicians (see Chapter 81). A fluores-
cence microscope is needed to read the IFA slides; therefore, this
assay is usually only performed in diagnostic laboratories. If the
specimens cannot be examined immediately, they can be stored
at 4°C for a maximum of several days but should not be frozen.
Fecal Antigen Detection by Enzyme-Linked Immunosorbent Assays
Several ELISAs are available for detection of Giardia antigen
in human feces. A veterinary assay is labeled for use with dog
and cat feces (SNAP Giardia, IDEXX Laboratories, Portland,
ME). Results vary with the ELISA assay used, and inconsis-
tent results have been reported with some Giardia antigen tests
Performance
False negatives are common and occur when specimen
size is small or low numbers of organisms are present,
or because of intermittent shedding. False positives
can occur when cysts are confused with yeasts.
Because of the intermittent pattern of shedding, at least
three specimens should be examined over a period of
about 1 week before concluding that Giardia spp. in-
fection is an unlikely cause of diarrhea. False positives
can occur when cysts are confused with yeasts.
May be more sensitive and specific than centrifugal fecal
flotation, but sensitivity for detection of assemblages
that infect dogs and cats is not well understood.
Sensitivity appears to be similar to that of Giardia
antigen ELISA assays.
When used in combination with centrifugal fecal flota-
tion, sensitivity can approach 98%. However, perfor-
mance varies with the assay used, and the sensitivity
of ELISA assays for detection of assemblages from
dogs and cats is not well understood. It has been
recommended that this assay be used in addition to
fecal flotation only during evaluation of dogs and
cats with diarrhea and not healthy pets (see text).
The sensitivity and specificity of assays may vary
depending on assay design. False negatives can occur
as a result of PCR inhibitors in feces. The significance
of a positive PCR result must be interpreted in light
of clinical findings because of subclinical infections.
when compared to other techniques. In a study of cats, the
SNAP Giardia antigen assay had similar sensitivity and specific-
ity when compared to fecal flotation, and when the results of
the two techniques were combined, the overall sensitivity was
97.8%.14 Little information is available as to whether currently
available fecal antigen assays detect Giardia assemblages C, D,
or F (i.e., the canine- and feline-associated assemblages). In one
study of 4 assemblage C isolates and 13 assemblage D isolates
(which infect dogs), all were positive by the SNAP Giardia anti-
gen assay.15 The Companion Animal Parasite Council currently
recommends that fecal antigen tests be used as an addition to
fecal flotation only during the evaluation of dogs and cats with
diarrhea and not healthy pets, because the clinical and zoo-
notic impact of an antigen-positive, cyst-negative healthy pet
is unknown.16 It is also unknown how long Giardia antigen
assays remain positive after resolution of diarrhea. Therefore, if
a veterinarian chooses to assess the success of the treatment for
giardiasis in cats and dogs, only fecal flotation is recommended
for the follow-up evaluation.
Molecular Diagnosis Using the Polymerase Chain Reaction
Giardia spp. DNA can be amplified from feces by PCR assay,
and such molecular methods can be used to determine the
Giardia assemblage present through sequence analysis of the
PCR product. However, the assignment of isolates to specific
G. duodenalis assemblages using PCR assays is not always

consistent because different results occur depending on the
target sequence amplified.5,17 Thus, some dog or cat isolates
can be genotyped as “potentially zoonotic” using one target
gene but as “host specific” with another. It is currently recom-
mended that if the assemblage is to be determined, three genes
should be assessed (the assessment of multiple genes using PCR
and sequencing is also known as multilocus sequence typing).
Giardia PCR assays can also be falsely negative because of the
presence of PCR inhibitors in feces and so should not be used
as the sole Giardia spp. assay.
Pathologic Findings
Gross Pathologic Findings
Giardia spp. infection may cause mild intestinal thickening.
Because the agent is not enteroinvasive, blood is generally not
present within the intestinal lumen unless co-infections are rec-
ognized. Animals with chronic clinical signs of disease may be
emaciated. Other parasites such as roundworms or hookworms
may be found.
Histopathologic Findings
In clinically affected animals with Giardia infection, a diffuse
loss of brush border microvillus length may be noted on histo-
pathology of the small intestine. Increased numbers of intraepi-
thelial lymphocytes and mast cell hyperplasia may also be
present. However, diarrhea has been associated with giardiasis
in dogs, cats, and people with normal intestinal biopsy results.
Treatment and Prognosis
Giardia spp. have specific antimicrobial susceptibility patterns,
and so it is currently impossible to predict which anti-Giardia
drug will be effective in individual dogs or cats. Because Giardia
spp. of dogs and cats can be difficult to culture in the laboratory,
there is little in vitro susceptibility test result information avail-
able. Although a variety of drugs have been used to treat giardia-
sis in dogs and cats, there are few studies that have evaluated the
efficacy of drugs and varied drug doses in experimentally infected
animals. In most studies, fecal specimens were evaluated for only
short periods of time after treatment, and immune suppression
was not induced to evaluate whether infection was eliminated
or merely suppressed. Infection with Giardia does not appear to
cause permanent immunity, and so re-infection can occur, a find-
ing that also hampers assessment of treatment studies.
Treatment options that are currently available or used his-
torically for dogs or cats with confirmed or suspected giardiasis
include albendazole, febantel/pyrantel/praziquantel, fenbenda-
zole, furazolidone, ipronidazole, metronidazole, quinacrine,
and tinidazole (Table 79-3). Newer drugs being studied or those
with minimal information available for dogs and cats include
azithromycin, nitazoxanide, paromomycin, and secnidazole.
Administration of metronidazole is indicated if concurrent
infection with Clostridium spp. is suspected (see Chapter 48). In
cats, metronidazole benzoate is preferred to metronidazole USP
because it is better tolerated by most cats.18 However, metroni-
dazole should not be administered orally for longer than 7 days
sequentially or at a total daily dose greater than 50 mg/kg because
of the risk for neurotoxicity.19,20 Ipronidazole, ronidazole, secni-
dazole, and tinidazole are other drugs with this class that have
been used to treat giardiasis in dogs or cats (see Table 79-3). Ipro-
nidazole added to the drinking water could be considered if a large
number of dogs are clinically affected.21 Ronidazole was used
CHAPTER 79  Giardiasis 775
TABLE 79-3
Drugs Used for the Treatment of Giardia spp. Infections
Drug Species Dose
Febantel, pyrantel, D Label dose for 3 days
praziquantel C 56 mg/kg (based on the febantel
component) PO q24h for 5
days
Fenbendazole D, C 50 mg/kg PO daily for
3-5 days
Ipronidazole D 126 mg/liter of water PO ad
libitum for 7 days
Metronidazole D, C 15-25 mg/kg PO q12-24h for
5-7 days
Tinidazole D 44 mg/kg PO q24hr for
3 days
C 30 mg/kg PO q24hr for
3 days
C, Cat; D, dog.
with hygiene management to control giardiasis in a dog kennel,
but the author recommends reserving this drug for the treatment
of T. foetus infections because of the potential for neurotoxicity
and emerging ronidazole-resistant T. foetus strains.22-24 Tinida-
zole should be considered as a secondary Giardia spp. treatment
for dogs and cats if metronidazole is not tolerated, but optimal
dosing protocols are not known. Secnidazole was evaluated in
a small study of cats with minimal recognized toxicity (elevated
liver enzyme activity in one cat) and apparent efficacy after one
dose.25 Further research is needed before routine use of this drug
can be recommended.
If concurrent nematode infestation is suspected, albendazole,
febantel/pyrantel/praziquantel, or fenbendazole may be most
effective anti-Giardia drugs.26-35 Albendazole has been associ-
ated with bone marrow suppression in some dogs and cats, so
some clinicians avoid use of this drug.35 Febantel/pyrantel/pra-
ziquantel is licensed in some countries for treatment of Giardia
spp. infection in dogs and can be effective when administered
daily for 3 days.26 This combination has also been studied in
small numbers of experimentally infected cats.33 Data from
an experimental model suggest that the combination of feb-
antel with pyrantel is synergistic.34 Many clinicians currently
prescribe fenbendazole once daily for 5 days as initial therapy
for dogs or cats with diarrhea and Giardia infection because of
minimal expense and wide safety margin.
If the first drug fails to clear the infection (cyst shedding)
or resolve the diarrhea, antimicrobial drug resistance may be
present, so a second drug from an alternative class is indicated.
Some clinicians currently recommend the concurrent admin-
istration of metronidazole and fenbendazole. Other clinicians
resort to combination therapy only if there is evidence of a
persistent infection that has not been cleared by monotherapy.
Azithromycin was used successfully in the management of
giardiasis in one dog.36 Paromomycin has anti-Giardia proper-
ties but has been absorbed across the gastrointestinal epithe-
lium and associated with reversible acute renal failure in cats.
Nitazoxanide is labeled for the treatment of giardiasis and cryp-
tosporidiosis in humans in some countries and has been studied
776 SECTION 4  Protozoal Diseases
in some dogs and cats, but optimal protocols for the treatment
of giardiasis are unknown. In addition, nitazoxanide is com-
monly associated with vomiting in dogs and cats.31 The author
currently administers nitazoxanide at 10 mg/kg PO q12h with
food for 7 days for the treatment of dogs or cats with small
bowel diarrhea and co-infections with Giardia spp. and Cryp-
tosporidium spp. A longer duration of treatment appears to
be required for some dogs and cats, and so treatment should
be continued if an initial positive response is noted but clinical
resolution is not achieved after the first 7 days.
The addition of fiber to the diet may help control clinical
signs of giardiasis in some animals through reduction of bacterial
overgrowth or by inhibiting organism attachment to microvilli.
Feeding a fat-restricted diet may also be effective. In one study,
administration of the probiotic Enterococcus faecium SF68
(FortiFlora, Nestle Purina PetCare) lessened Giardia spp. shed-
ding in mice when fed before infection and enhanced Giardia
immune responses.37 In contrast, administration of this probi-
otic to dogs with chronic subclinical giardiasis had no measur-
able effect on cyst shedding.38 In another study, dogs with acute
diarrhea that were housed in shelters responded more rapidly to
treatment if the probiotic was administered with metronidazole
than if metronidazole was administered alone; many of these
dogs were infected with Giardia spp.39
Bathing all dogs during medical treatment may lessen
re-infection rates. In dogs and cats with persistent diarrhea
and Giardia spp. infection, a more extensive work-up to
attempt to diagnose other underlying diseases is indicated if
several therapeutic trials fail to control the diarrhea. Poten-
tial underlying disorders that should be considered include
cryptosporidiosis, tritrichomoniasis (primarily in cats),
inflammatory bowel disease, gastrointestinal lymphoma,
bacterial overgrowth, exocrine pancreatic insufficiency, and
immunodeficiencies.
The primary goal of Giardia treatment is to resolve clini-
cal signs of diarrhea. It is controversial whether to treat
healthy dogs and cats with Giardia spp. cysts in feces,
because healthy pets are generally not considered significant
human health risks by the Centers for Disease Control and
Prevention.40 This is because infection can recur rapidly or
not be cleared, and because all drugs have adverse effects.
For example, in one small study of naturally infected dogs,
approximately 50% of dogs had adverse events associated
with treatment, and more than 60% of treated dogs were still
Giardia infected when rechecked 34 days after treatment.31
However, because clinical signs induced by Giardia spp. can
be intermittent and since some Giardia spp. may be of zoo-
notic concern (especially to the immunocompromised), treat-
ment of healthy infected animals should be discussed with
each owner. If treatment is deemed indicated by the clini-
cian and owner, many clinicians currently recommend that a
5-day course of fenbendazole be administered to apparently
healthy dogs and cats that test positive for Giardia spp. cysts
in feces. After treatment, if the animal is healthy and nega-
tive for cysts, retesting is not indicated again until the next
scheduled wellness examination and fecal flotation. Giardia–
infected dogs or cats should not be tested for Giardia antigen
or DNA in feces since it is unknown how long positive assay
results persist after treatment.
A common question is how to manage dogs or cats that
have normal stool and are Giardia antigen positive but Giardia
cyst negative. These animals may have a low-grade infection,
or a low percentage of animals (approximately 2% to 5%)
have false-positive antigen test results. To further evaluate for
cyst shedding, the veterinarian can perform an IFA test or two
additional fecal flotations (three negative centrifugal flotation
assays run within 5 days is considered adequate to rule out a
Giardia infection in both animals and humans). If these other
test results are negative, the antigen test was likely falsely posi-
tive. If cysts are still identified after fecal centrifugal flotation
performed 2 weeks after appropriate administration of a drug
with anti-Giardia activity, a second course of therapy may be
indicated using a drug from a different class. It is recommended
that apparently well animals not be treated beyond two courses
of therapy.40 No further diagnostics are indicated unless gas-
trointestinal signs occur or the animal is again due for routine
fecal screening (once to twice annually as a minimum for all
dogs and cats).
Immunity and Vaccination
Sterilizing immunity to Giardia spp. does not occur, and so
repeated shedding, clinical signs, and infection may occur. Re-
infection occurs even in vaccinated dogs or cats, and so the
previously available Giardia vaccines have been discontinued.
Prevention
Prevention of Giardia infection involves boiling or filtering
of water collected from the environment before drinking and
disinfection of premises contaminated with infected feces with
steam cleaning or quaternary ammonium compounds (1 minute
contact time). Transport hosts should be controlled, and treat-
ment and bathing of all animals in the environment could be
considered if intermittent diarrhea occurs. Feces from infected
animals should be removed from the environment promptly.
Public Health Aspects
In humans, infection with Giardia can be asymptomatic or
result in acute or chronic gastrointestinal illness. Acute giar-
diasis is characterized by watery diarrhea, epigastric pain,
nausea, vomiting, decreased appetite, and weight loss; these
signs occur 6 to 15 days after infection. Young children and
immunodeficient individuals are most likely to show clinical
signs; chronic infections are also more likely to occur in the
immunocompromised. Recent studies suggest that functional
gastrointestinal disorders such as irritable bowel syndrome can
be associated with a previous Giardia duodenalis infection.41
Infection with the Giardia assemblages of dogs (assem-
blage C and D) or cats (assemblage F) is usually not associ-
ated with clinical disease in humans, but can be detected in the
feces of immunocompromised people.1 Human assemblages A
and B can be found in feces of dogs and cats when animals
share human environmental features (i.e., contaminated water).
However, it is unlikely that new human infections by Giardia
assemblages A or B are acquired from dogs or cats.3 Thus,
healthy animals that test positive for Giardia are probably not
significant human health risks if the family members are also
healthy. Greater risk is likely to exist for immunocompromised
people, and so the potential for contact with infected feces
should be minimized.

CASE EXAMPLE
Signalment: “Duke,” a 12-week-old intact male Golden
retriever from Fort Collins, CO
History: Duke was adopted from a backyard breeder.
The puppy was eating well and was in good body
condition. Small bowel diarrhea was the only historical
complaint. The stool character varied from soft to
watery with occasional streaks of white material that
was interpreted as mucus. The diarrhea began just after
adoption (a week before evaluation). This was also the
time that a new commercial puppy food designed for
large-breed dogs was introduced by the owner. The
puppy’s first two vaccines (attenuated live distemper,
adenovirus, parvovirus, parainfluenza virus) had been
administered at 7 and 10 weeks of age, at which times
he also was administered pyrantel pamoate orally for
strategic deworming. It was unknown whether or not
other littermates had diarrhea or parasitic infections. The
resident adult collie was considered normal by the owner,
and the adult dog’s feces were normal the previous day.
Physical Examination: The dog was bright, alert, and
responsive and weighed 11.2 kg. T = 101.3°F (38.5°C), HR =
120 beats/min, RR = 24 breaths/min, mucous membranes
pink, and CRT = 1 s. Body condition score was 4/9. The
dog’s haircoat was normal and there was no evidence of
ectoparasites. No clinically significant abnormalities were
noted on physical examination.
Results of Microbiologic Testing: Fecal wet mount
examination: No motile flagellates were noted.
Zinc sulfate centrifugal fecal flotation: cysts consistent with
Giardia spp. were noted.
Fecal cytology: no inflammatory cells, spore-forming rods, or
spirochetes were detected.
Diagnosis: Giardia spp. infection.
SUGGESTED READINGS
Scorza V, Lappin MR. Giardiasis. In: Greene CE, ed. Infectious Diseases
of the Dog and Cat. 4th ed. St. Louis, MO: Elsevier; 2012:785-792.
Simpson KW, Rishniw M, Bellosa M, et  al. Influence of Enterococ-
cus faecium SF68 probiotic on giardiasis in dogs. J Vet Intern Med.
2009;23:476-481.
Thompson RCA, Smith A. Zoonotic enteric protozoa. Vet Parasitol.
2011;182(1):70-78.
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3. Scorza V, Lappin MR. Giardiasis. In: Greene CE, ed. Infectious
Diseases of the Dog and Cat. 4th ed. St. Louis, MO: Elsevier;
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4. Kirkpatrick CE, Farrell JP. Feline giardiasis: Observations on natural
and induced infections. Am J Vet Res. 1984;45:2182-2188.
CHAPTER 79  Giardiasis 777
Treatment: Fenbendazole (50 mg/kg PO q24h) for 3 days
was prescribed together with a commercially available
intestinal diet and probiotic (daily for 1 month). The
adult housemate was not treated. The feces were normal
2 days later, after which the puppy food was reinstituted by
feeding a progressively increasing percentage over 5 days.
Clinical signs of diarrhea never developed in the adult dog.
A second vaccine was administered at the recheck 1 week
later and again with a rabies vaccine at 16 weeks of age, at
which time a fecal flotation was negative. The puppy was
still clinically healthy at the 16-week vaccine visit.
Comments: The findings were characteristic of Giardia spp.–
associated diarrhea; however, because Giardia spp. cysts are
common in both healthy puppies and puppies with diarrhea,
it is impossible to definitively make a diagnosis of giardiasis.
Since the puppy needed an additional strategic deworming,
fenbendazole was chosen rather than metronidazole.
Febantel/praziquantel/pyrantel would also have been an
appropriate choice. Because the history was also consistent
with stress-associated diarrhea (diet change, housing
change), the probiotic and diet change were initiated. Both
of these adjunct treatments also may aid in the treatment
of giardiasis. Giardia antigen testing was not performed,
because cysts were clearly identified on fecal flotation.
Giardia assemblage determination was not performed,
but could have been considered if immunosuppressed
family members lived in the household. If recheck fecal
examination is to be performed, only fecal flotation is
indicated, not fecal antigen tests or PCR assays. Whether to
test or treat in-contact healthy adult dogs is controversial;
however, 5% to 10% of healthy dogs are already infected
with Giardia in most studies, re-infection is common, and
drugs are expensive and can be toxic. Thus, many clinicians
just treat the animal with clinical signs of disease during
the first episode of diarrhea, with the potential to escalate
therapies should the problem persist.
6. Scorza AV, Ballweber LR, Tangtrongsup S, et  al. Comparisons
of mammalian Giardia duodenalis assemblages based on the
β-giardin, glutamate dehydrogenase and triose phosphate isomerase
genes. Vet Parasitol. 2012;189:182-188.
7. Weissenböck H, Ondrovics M, Gurtner S, et al. Development of a
chromogenic in situ hybridization for Giardia duodenalis and its
application in canine, feline, and porcine intestinal tissue samples.
J Vet Diagn Invest. 2011;23:486-491.
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9. Buret AG. Pathophysiology of enteric infections with Giardia duo-
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11. Gookin JL, Stebbins ME, Hunt E